Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof

ABSTRACT

The invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator (uPA), to activatable antibodies and other larger molecules that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator, and to methods of making and using these polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator in a variety of therapeutic, diagnostic and prophylactic indications.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/610,468, filed Jan. 30, 2015, which claims the benefit of U.S.Provisional Application No. 61/934,619, filed Jan. 31, 2014 and U.S.Provisional Application No. 61/971,009, filed Mar. 27, 2014, thecontents of each of which are incorporated herein by reference in theirentireties.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “CYTM034D01USSeqList.txt,” which wascreated on Dec. 27, 2016 and is 360 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to polypeptides that include a cleavablemoiety that is a substrate for at least one protease selected frommatriptase and u-plasminogen activator (uPA), to activatable antibodiesand other larger molecules that include the cleavable moiety that is asubstrate for at least one protease selected from matriptase and uPA,and to methods of making and using these polypeptides that include acleavable moiety that is a substrate for at least one protease selectedfrom matriptase and uPA in a variety of therapeutic, diagnostic andprophylactic indications.

BACKGROUND OF THE INVENTION

Proteases are enzymes that cleave the peptide bonds between amino acidresidues. Some proteases are known to break specific peptide bonds basedon the presence of a particular amino acid sequence within a protein.Proteases occur naturally in all organisms and are involved in a varietyof physiological reactions from simple degradation to highly regulatedpathways. However, many pathological conditions are associated withderegulated expression and/or activity of proteases. As such,inappropriate proteolysis can have a major role in development andprogression of cancer as well as cardiovascular, inflammatory,neurodegenerative, eukaryotic, bacterial and viral and parasiticdiseases.

Accordingly, there exists a need to identify new substrates forproteases and to use these substrates in a variety of therapeutic,diagnostic and prophylactic indications.

SUMMARY OF THE INVENTION

The disclosure provides amino acid sequences that include a cleavablemoiety (CM) that is a substrate for at least one protease selected frommatriptase (also referred to herein as MT-SP1, matriptase-1, and similarterms denoting matriptase) and u-plasminogen activator (also referred toherein as uPA, urokinase, urokinase-type plasminogen activator, andsimilar terms denoting uPA). These CMs are useful in a variety oftherapeutic, diagnostic and prophylactic indications.

In some embodiments, the CM is linked or otherwise attached to anantibody. For example, the CM is used to link one or more agents to theantibody or antigen binding fragment thereof that binds a given target,such that the CM is cleaved when exposed to the protease, i.e.,matriptase and/or uPA, and the agent is released from the antibody orantigen-binding fragment. Exemplary targets include, but are not limitedto the targets shown in Table 1. Exemplary antibodies or antigen-bindingfragments thereof include, but are not limited to, the targets shown inTable 2. In some embodiments, the antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:Agent-CM-(Antibody or Antigen-Binding Fragment) or (Antibody orAntigen-Binding Fragment)-CM-Agent. In some embodiments, the antibodycomprises a linking peptide between the antibody or antigen-bindingfragment and the CM. In some embodiments, the antibody orantigen-binding fragment comprises a linking peptide between the CM andthe conjugated agent.

In some embodiments, the antibody comprises a first linking peptide(LP1) and a second linking peptide (LP2), wherein the antibody in theuncleaved state has the structural arrangement from N-terminus toC-terminus as follows: Agent-LP1-CM-LP2—(Antibody or Antigen-BindingFragment) or (Antibody or Antigen-Binding Fragment)-LP2-CM-LP1-Agent. Insome embodiments, the two linking peptides need not be identical to eachother.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 385) and (GGGS)_(n) (SEQ ID NO: 386), where n isan integer of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 387),GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO: 389), GSGGG (SEQ ID NO: 390),GGGSG (SEQ ID NO: 391), and GSSSG (SEQ ID NO: 392).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 393), GSSGGSGGSGG (SEQ ID NO: 394), GSSGGSGGSGGS (SEQ ID NO:395), GSSGGSGGSGGSGGGS (SEQ ID NO: 396), GSSGGSGGSG (SEQ ID NO: 397), orGSSGGSGGSGS (SEQ ID NO: 398).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS,GGGS (SEQ ID NO: 399), GSSGT (SEQ ID NO: 400) or GSSG (SEQ ID NO: 401).

In some embodiments, the antibody or antigen-binding fragment has anequilibrium dissociation constant of about 100 nM or less for binding tothe target.

In some embodiments, the antibody or antigen-binding fragment thereofspecifically binds a target. In some embodiments, the antibody orimmunologically active fragment thereof that binds the target is amonoclonal antibody, domain antibody, single chain, Fab fragment, aF(ab′)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chainantibody, or a single domain light chain antibody. In some embodiments,such an antibody or immunologically active fragment thereof that bindsthe target is a mouse, other rodent, chimeric, humanized or fully humanmonoclonal antibody.

In some embodiments, the protease, i.e., matriptase and/or uPA isco-localized with the target in a tissue, and the protease cleaves theCM in the antibody when the antibody is exposed to the protease.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM is a substrate for at least matriptase. Insome embodiments, the CM is a substrate for at least uPA. In someembodiments, the CM is a substrate for at least matriptase and uPA.

In some embodiments, the CM is a substrate for matriptase and/or uPA,and is resistant to cleavage by at least one other protease. In someembodiments, the CM is a substrate for matriptase and/or uPA, and isresistant to cleavage by at least plasmin. In some embodiments, the CMis a substrate for matriptase and/or uPA, and is resistant to cleavageby at least tissue plasminogen activator (tPA).

In some embodiments, the CM is a substrate for matriptase and/or uPA andincludes a motif sequence that is recognized by matriptase and/or uPA,provided that for any given motif sequence of the disclosure:

-   -   (i) the CM does not comprise any of the following amino acid        sequences TGRGPSWV (SEQ ID NO: 402), SARGPSRW (SEQ ID NO: 403),        or TARGPSFK (SEQ ID NO: 404); and the CM does not comprise a        consensus amino acid sequence based on these amino acid        sequences, such as for example, TARGPSW (SEQ ID NO: 405);    -   (ii) the CM does not comprise any of the following amino acid        sequences LSGRSDNH (SEQ ID NO: 406), GGWHTGRN (SEQ ID NO: 407),        HTGRSGAL (SEQ ID NO: 408), or PLTGRSGG (SEQ ID NO: 409); and the        CM does not comprise a consensus amino acid sequence based on        these amino acid sequences, such as for example, LTGRSGA (SEQ ID        NO: 410); and/or    -   (iii) the CM does not comprise any of the following amino acid        sequences AARGPAIH (SEQ ID NO: 411), RGPAFNPM (SEQ ID NO: 412),        SSRGPAYL (SEQ ID NO: 413), or RGPATPIM (SEQ ID NO: 414); and the        CM does not comprise a consensus amino acid sequence based on        these amino acid sequences, such as for example, RGPA (SEQ ID        NO: 415).

In some embodiments, the motif sequence is a substrate for at leastmatriptase and includes a core CM consensus sequence shown in Tables8A-8J below. In some embodiments, the motif sequence includes asubgenus, i.e., a subset, of the core CM consensus sequence shown inTables 8A-8J below.

TABLE 8A Matriptase Cleavable Core CM Consensus Sequence 1 Core CMConsensus 1 Subgenus of Core CM Consensus 1 X₁X₂X₃X₄X₅ (SEQ ID NO: 1),wherein: X₁X₂X₃X₄X₅ (SEQ ID NO: 2), wherein X₁ is A, X₁ is A, G, H, K,L, N, P, R, S, or V; G, P, R, S, or V; X₂ is A, L, M, P, S, or V; X₃ isX₂ is A, H, L, M, P, Q, R, S, or V; G, L, or P; X₄ is R; and X₅ is A, G,R, S, or V X₃ is A, E, F, G, I, L, P, R, S, T, or V; X₁X₂X₃X₄X₅ (SEQ IDNO: 3), wherein X₁ is A, X₄ is A, I, K, N, R, T, or W; and P, R, S, orV; X₂ is A, L, M, S, or V; X₃ is P; X₄ X₅ is A, G, I, L, M, Q, R, S, orV is R; and X₅ is A, G, S, or V X₁X₂X₃X₄X₅ (SEQ ID NO: 4), wherein: X₁is A, P, or R; X₂ is A, S, or V; X₃ is P; X₄ is R; and X₅ is S or VX₁X₂X₃X₄X₅ (SEQ ID NO: 5), wherein: X₁ is A, P, or R; X₂ is A or S; X₃is P; X₄ is R; and X₅ is S

TABLE 8B Matriptase Cleavable Core CM Consensus Sequence 2 Core CMConsensus 2 Subgenus of Core CM Consensus 2 X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO:6), wherein: X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 7), wherein: X₁₀ X₁₀ is A, L,P, R, S, T, or V; is A, R, S, or T; X₁₁ is K or R; X₁₂ is D or R; X₁₃X₁₁ is K or R; is L or V; and X₁₄ is F or P X₁₂ is D or R;X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 8), wherein: X₁₀ X₁₃ is A, G, L, M, S, T, V,or W; and is A, S, or T; X₁₁ is K or R; X₁₂ is R; X₁₃ is L or X₁₄ is F,G, M, P, or V V; and X₁₄ is F or P X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 9),wherein: X₁₀ is S or T; X₁₁ is R; X₁₂ is R; X₁₃ is L or V; and X₁₄ is P

TABLE 8C Matriptase Cleavable Core CM Consensus Sequence 3 Core CMConsensus 3 Subgenus of Core CM Consensus 3 X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO:10), wherein: X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 11), wherein: X₂₀ is E, G, P,R, S, V, or W; X₂₀ is G, P, R, S, or V; X₂₁ is P or V; X₂₂ is L or X₂₁is A, G, L, M, P, S, or V; R; X₂₃ is G; and X₂₄ is G or R X₂₂ is A, I,L, or R; X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 12), wherein: X₂₃ is A, G, I, or P;and X₂₀ is P or R; X₂₁ is P; X₂₂ is L; X₂₃ is G; and X₂₄ is G or R X₂₄is R

TABLE 8D Matriptase Cleavable Core CM Consensus Sequence 4 Core CMConsensus 4 Subgenus of Core CM Consensus 4 X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO:13), wherein: X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 14), wherein: X₂₆ is A, G, H,L, R, or S; X₂₆ is A, G, H, L, R, or S; X₂₇ is D, H, R, or T; X₂₇ is D,H, N, R, S, T, or W; X₂₈ is A, P, R, S, T, or V; X₂₉ is F, G, L, M, P,or X₂₈ is A, N, P, R, S, T, or V; S; and X₃₀ is G, L, P, S, V, or W X₂₉is F, G, L, M, P, Q, R, S, or Y; and X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 15),wherein: X₃₀ is I, G, L, P, S, V, or W X₂₆ is G, L, or S; X₂₇ is R or T;X₂₈ is A, P, or S; X₂₉ is F, G, M, or S; and X₃₀ is G, P, S, V, or WX₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 16), wherein: X₂₆ is G, L, or S; X₂₇ is R;X₂₈ is A or S; X₂₉ is G or M; and X₃₀ is G, P, S, or W X₂₆X₂₇X₂₈X₂₉X₃₀(SEQ ID NO: 17), wherein: X₂₆ is L; X₂₇ is R; X₂₈ is A or S; X₂₉ is G;and X₃₀ is W

TABLE 8E Matriptase Cleavable Core CM Consensus Sequence 5 Core CMConsensus 5 Subgenus of Core CM Consensus 5 X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO:18), wherein: X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 19), wherein: X₃₆ is G, K, L,S, V, or W; X₃₆ is G, L, S, V, or W; X₃₇ is G, Q, R, or S; X₃₈ X₃₇ is G,I, P, Q, R, or S; is R; X₃₉ is G, S, or V; and X₄₀ is A, G, L, S, and VX₃₈ is R; X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 20), wherein: X₃₉ is G, K, R, S,or V; and X₃₆ is V; X₃₇ is S; X₃₈ is R; X₃₉ is S; and X₄₀ is X₄₀ is A,C, G, L, M, P, S, V, or Y A and V

TABLE 8F Matriptase Cleavable Core CM Consensus Sequence 6 Core CMConsensus 6 Subgenus of Core CM Consensus 6 X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO:21), wherein: X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 22), wherein: X₄₂ is A, E, G,I, L, M, R, or S; X₄₂ is A, E, G, L, M, R, or S; X₄₃ is G, K, L, N, X₄₃is A, G, K, L, N, R, S, or V; R, S, or V; X₄₄ is R or Y; X₄₅ is A, F, G,P, or S; X₄₄ is F, H, L, R, or Y; and X₄₆ is F, G, M, P, R, S, or V X₄₅is A, F, G, H, P, or S; and X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 23), wherein:X₄₆ is F, G, M, N, P, R, S, or V X₄₂ is A, E, G, M, or S; X₄₃ is G, L,S, or V; X₄₄ is R or Y; X₄₅ is A, G, P, or S; and X₄₆ is F, G, M, P, R,S, or V X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 24), wherein: X₄₂ is A, G, or S; X₄₃is L, S, or V; X₄₄ is R; X₄₅ is A; and X₄₆ is M or P X₄₂X₄₃X₄₄X₄₅X₄₆(SEQ ID NO: 25), wherein: X₄₂ is A; X₄₃ is L, S or V; X₄₄ is R; X₄₅ isA; and X₄₆ is M or P

TABLE 8G Matriptase Cleavable Core CM Consensus Sequence 7 Core CMConsensus 7 Subgenus of Core CM Consensus 7 X₅₀X₅₁X₅₂X₅₃X₅₄ (SEQ ID NO:26), wherein: X₅₀X₅₁X₅₂X₅₃X₅₄ (SEQ ID NO: 27), wherein: X₅₀ is A, E, K,L, P, S, T, V, W, or Y; X₅₀ is E, P, S, V, or W; X₅₁ is A, P, R, S, V,or X₅₁ is A, I, L, P, R, S, V, or Y; Y; X₅₂ is E, G, H, L, P, or V; X₅₃is G, K, L, or X₅₂ is E, G, H, L, P, or V; R; and X₅₄ is Q or R X₅₃ isG, K, L, or R; and X₅₀X₅₁X₅₂X₅₃X₅₄ (SEQ ID NO: 28), wherein: X₅₄ is Q orR X₅₀ is P or V; X₅₁ is A, P, or R; X₅₂ is E, G, P, or V; X₅₃ is G or R;and X₅₄ is R X₅₀X₅₁X₅₂X₅₃X₅₄ (SEQ ID NO: 29), wherein: X₅₀ is P or V;X₅₁ is A or R; X₅₂ is G or V; X₅₃ is G or R; and X₅₄ is RX₅₀X₅₁X₅₂X₅₃X₅₄ (SEQ ID NO: 30), wherein: X₅₀ is P or V; X₅₁ is A; X₅₂is G or V; X₅₃ is R; and X₅₄ is R

TABLE 8H Matriptase Cleavable Core CM Consensus Sequence 8 Core CMConsensus 8 Subgenus of Core CM Consensus 8 X₅₇X₅₈X₅₉X₆₀X₆₁ (SEQ ID NO:31), wherein: X₅₇X₅₈X₅₉X₆₀X₆₁ (SEQ ID NO: 32), wherein: X₅₇ is A, G, I,K, P, S, or T; X₅₇ is A, G, I, K, S, or T; X₅₈ is R; X₅₉ is S; X₆₀ X₅₈is R or T; is F, M, or R; and X₆₁ is A, I, L, R, or W X₅₉ is H, M, or S;X₅₇X₅₈X₅₉X₆₀X₆₁ (SEQ ID NO: 33), wherein: X₆₀ is F, M, or R; and X₅₇ isG or K; X₅₈ is R; X₅₉ is S; X₆₀ is M; and X₆₁ is A, G, I, L, P, Q, R, S,V, or W X₆₁ is A, L, R, or W X₅₇X₅₈X₅₉X₆₀X₆₁ (SEQ ID NO: 34), wherein:X₅₇ is G; X₅₈ is R; X₅₉ is S; X₆₀ is M; and X₆₁ is A or L

TABLE 8I Matriptase Cleavable Core CM Consensus Sequence 9 Core CMConsensus 9 Subgenus of Core CM Consensus 9 X₆₇X₆₈X₆₉X₇₀X₇₁ (SEQ ID NO:35), wherein: X₆₇X₆₈X₆₉X₇₀X₇₁ (SEQ ID NO: 36), wherein: X₆₇ is I, L, orS; or V X₆₇ is I or L; X₆₈ is A, G, P, or V; X₆₉ is R; X₇₀ X₆₈ is A, G,K, P, R, or V; is A, M, P, R, S, or T; and X₇₁ is G, K, L, R, S, X₆₉ isL, R or S; X₆₇X₆₈X₆₉X₇₀X₇₁ (SEQ ID NO: 37), wherein: X₇₀ is A, K, M, P,R, S, or T; and X₆₇ is L; X₆₈ is A, G, P, or V; X₆₉ is R; X₇₀ is A, X₇₁is F, G, H, I, K, L, M, P, R, S, or V M, P, R, or S; and X₇₁ is G, K, L,S, or V X₆₇X₆₈X₆₉X₇₀X₇₁ (SEQ ID NO: 38), wherein: X₆₇ is L; X₆₈ is A, G,or P; X₆₉ is R; X₇₀ is A or S; and X₇₁ is G or L X₆₇X₆₈X₆₉X₇₀X₇₁ (SEQ IDNO: 39), wherein: X₆₇ is L; X₆₈ is A or P; X₆₉ is R; X₇₀ is A; and X₇₁is G or L

TABLE 8J Matriptase Cleavable Core CM Consensus Sequence 10 Core CMConsensus 10 Subgenus of Core CM Consensus 10 X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ IDNO: 40), wherein: X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ ID NO: 41), wherein: X₇₄ is E, L,Q, S, T or V; X₇₄ is E, L, T or V; X₇₅ is R or S; X₇₆ is K or R; X₇₅ isL, R, or S; X₇₇ is M, R, or S; and X₇₈ is G, L, M, S, or W X₇₆ is H, K,or R; X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ ID NO: 42), wherein: X₇₇ is A, M, R, or S;and X₇₄ is E, T or V; X₇₅ is R or S; X₇₆ is K or R; X₇₇ X₇₈ is G, L, M,R, S, or W is M or R; and X₇₈ is G, L, M, S, or W X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQID NO: 43), wherein: X₇₄ is E or V; X₇₅ is S; X₇₆ is K or R; X₇₇ is R;and X₇₈ is L, S, or W X₇₄X₇₅X₇₆X₇₇X₇₈ (SEQ ID NO: 44), wherein: X₇₄ isE; X₇₅ is S; X₇₆ is K or R; X₇₇ is R; and X₇₈ is L or W

In some embodiments, the motif sequence is a substrate for at leastmatriptase and includes an expanded consensus sequence based on one ofthe core CM consensus sequence shown in Tables 8A-8J. In someembodiments, the expanded consensus sequence is a consensus sequenceshown in Tables 9A-9J-3 below.

TABLE 9A Matriptase Cleavable Expanded Core CM Consensus Sequence 1Subgenus of Expanded Core CM Expanded Core CM Consensus 1 Consensus 1X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 45) X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 46), wherein: X₁is wherein: A, G, P, R, S, or V; X₂ is A, L, M, P, S, or V; X₃ is X₁ isA, G, H, K, L, N, P, R, S, or V; G, L, or P; X₄ is R; X₅ is A, G, R, S,or V; and X₆ X₂ is A, H, L, M, P, Q, R, S, or V; is F, G, H, L, M, S, orW, X₃ is A, E, F, G, I, L, P, R, S, T, or V; X₁X₂X₃X₄X₅X₆ (SEQ ID NO:47), wherein: X₁ is X₄ is A, I, K, N, R, T, or W; A, P, R, S or V; X₂ isA, L, M, S, or V; X₃ is P; X₅ is A, G, I, L, M, Q, R, S, or V; and X₄ isR; X₅ is A, G, S, or V; and X₆ is F, G, H, M, X₆ is F, G, H, L, M, R, S,or W S, or W X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 48), wherein: X₁ is A, P, or R; X₂is A, S, or V; X₃ is P; X₄ is R; X₅ is S or V; and X₆ is F, G, H, M, orS X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 49), wherein: X₁ is A, P, or R; X₂ is A or S;X₃ is P; X₄ is R; X₅ is S; and X₆ is F, G, H, or S

TABLE 9B-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 2ASubgenus of Expanded Core CM Expanded Core CM Consensus 2A Consensus 2AX₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 50), X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 51),wherein: X₉ wherein: is E, G, L, P, Q or S; X₁₀ is A, R, S, or T; X₁₁ isK or X₉ is A, E, G, L, P, Q, S, T or V; R; X₁₂ is D or R; X₁₃ is L or V;and X₁₄ is F or P X₁₀ is A, L, P, R, S, T, or V; X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQID NO: 52), wherein: X₉ X₁₁ is K or R; is E, L, P or Q; X₁₀ is A, S, orT; X₁₁ is K or R; X₁₂ X₁₂ is D or R; is R; X₁₃ is L or V; and X₁₄ is For P X₁₃ is A, G, L, M, S, T, V, or W; and X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO:53), wherein: X₉ X₁₄ is F, G, M, P, or V is E, P or Q; X₁₀ is S or T;X₁₁ is R; X₁₂ is R; X₁₃ is L or V; and X₁₄ is P

TABLE 9B-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 2BSubgenus of Expanded Core CM Expanded Core CM Consensus 2B Consensus 2BX₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 54), X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 55),wherein: X₁₀ wherein: is A, R, S, or T; X₁₁ is K or R; X₁₂ is D or R;X₁₃ is X₁₀ is A, L, P, R, S, T, or V; L or V; X₁₄ is F or P; and X₁₅ isG, L, S or V X₁₁ is K or R; X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 56), wherein:X₁₀ X₁₂ is D or R; is A, S, or T; X₁₁ is K or R; X₁₂ is R; X₁₃ is L orV; X₁₃ is A, G, L, M, S, T, V, or W; X₁₄ is F or P; and X₁₅ is G, L, Sor V X₁₄ is F, G, M, P, or V; and X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 57),wherein: X₁₀ X₁₅ is G, L, M, N, P, S, V, or Y is S or T; X₁₁ is R; X₁₂is R; X₁₃ is L or V; X₁₄ is P; and X₁₅ is L or V

TABLE 9B-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 2CSubgenus of Expanded Core CM Expanded Core CM Consensus 2C Consensus 2CX₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 59),wherein: NO: 58), wherein: X₉ is E, G, L, P, Q or S; X₁₀ is A, R, S, orT; X₁₁ is X₉ is A, E, G, L, P, Q, S, T or V; K or R; X₁₂ is D or R; X₁₃is L or V; X₁₄ is F or P; X₁₀ is A, L, P, R, S, T, or V; and X₁₅ is G,L, S or V X₁₁ is K or R; X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 60), wherein:X₁₂ is D or R; X₉ is E, L, P or Q; X₁₀ is A, S, or T; X₁₁ is K or R; X₁₃is A, G, L, M, S, T, V, or W; X₁₂ is R; X₁₃ is L or V; X₁₄ is F or P;and X₁₅ is G, X₁₄ is F, G, M, P, or V; and L, S or V X₁₅ is G, L, M, N,P, S, V, or Y X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅ (SEQ ID NO: 61), wherein: X₉ is E, Por Q; X₁₀ is S or T; X₁₁ is R; X₁₂ is R; X₁₃ is L or V; X₁₄ is P; andX₁₅ is L or V

TABLE 9C-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 3ASubgenus of Expanded Core CM Expanded Core CM Consensus 3A Consensus 3AX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 62), X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 63),wherein: wherein: X₁₉ is G, K, or S; X₂₀ is G, P, R, S, or V; X₂₁ is Por X₁₉ is D, G, K, S, T, or V; V; X₂₂ is L or R; X₂₃ is G; and X₂₄ is Gor R X₂₀ is E, G, P, R, S, V, or W; X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 64),wherein: X₂₁ is A, G, L, M, P, S, or V; X₁₉ is G or S; X₂₀ is P or R;X₂₁ is P; X₂₂ is L; X₂₃ is X₂₂ is A, I, L, or R; G; X₂₄ is R X₂₃ is A,G, I, or P; and X₂₄ is G or R

TABLE 9C-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 3BSubgenus of Expanded Core CM Expanded Core CM Consensus 3B Consensus 3BX₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 66),wherein: NO: 65), wherein: X₁₈ is C, G, or S; X₁₉ is G, K, or S; X₂₀ isG, P, R, S, X₁₈ is C, G, I, L or S; or V; X₂₁ is P or V; X₂₂ is L or R;X₂₃ is G; and X₂₄ X₁₉ is D, G, K, S, T, or V; is G or R X₂₀ is E, G, P,R, S, V, or W; X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 67), wherein: X₂₁ is A,G, L, M, P, S, or V; X₁₈ is C, G, or S; X₁₉ is G or S; X₂₀ is P or R;X₂₁ is X₂₂ is A, I, L, or R; P; X₂₂ is L; X₂₃ is G; X₂₄ is R X₂₃ is A,G, I, or P; and X₂₄ is G or R

TABLE 9C-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 3CSubgenus of Expanded Core CM Expanded Core CM Consensus 3C Consensus 3CX₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO:69), NO: 68), wherein: wherein: X₁₇ is G or S; X₁₈ is C, G, or S; X₁₉ isG, X₁₇ is G or S; K, or S; X₂₀ is G, P, R, S, or V; X₂₁ is P or V; X₂₂is X₁₈ is C, G, I, L or S; L or R; X₂₃ is G; and X₂₄ is G or R X₁₉ is D,G, K, S, T, or V; X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄ (SEQ ID NO: 70), X₂₀ is E, G,P, R, S, V, or W; wherein: X₁₇ is G or S; X₁₈ is C, G, or S; X₁₉ is G orX₂₁ is A, G, L, M, P, S, or V; S; X₂₀ is P or R; X₂₁ is P; X₂₂ is L; X₂₃is G; X₂₄ is R X₂₂ is A, I, L, or R; X₂₃ is A, G, I, or P; and X₂₄ is Gor R

TABLE 9D-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 4ASubgenus of Expanded Core CM Expanded Core CM Consensus 4A Consensus 4AX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 71), X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 72),wherein: wherein: X₂₅ is G, M, R, or S; X₂₆ is A, G, H, L, R, or S; X₂₇X₂₅ is G, M, R, or S; is D, H, R, or T; X₂₈ is A, P, R, S, T, or V; X₂₉is F, X₂₆ is A, G, H, L, R, or S; G, L, M, P, or S; and X₃₀ is G, L, P,S, V, or W X₂₇ is D, H, N, R, S, T, or W; X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO:73), wherein: X₂₈ is A, N, P, R, S, T, or V; X₂₅ is G, M, R, or S; X₂₆is G, L, or S; X₂₇ is R or T; X₂₉ is F, G, L, M, P, Q, R, S, or Y; X₂₈is A, P, or S; X₂₉ is F, G, M, or S; and X₃₀ is G, and P, S, V, or W X₃₀is I, G, L, P, S, V, or W X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 74), wherein:X₂₅ is G, M, R, or S; X₂₆ is G, L, or S; X₂₇ is R; X₂₈ is A or S; X₂₉ isG or M; and X₃₀ is G, P, S, or W X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀ (SEQ ID NO: 75),wherein: X₂₅ is M; X₂₆ is L; X₂₇ is R; X₂₈ is A or S; X₂₉ is G; and X₃₀is W

TABLE 9D-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 4BSubgenus of Expanded Core CM Expanded Core CM Consensus 4B Consensus 4BX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁ (SEQ ID X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁ (SEQ ID NO: 77),wherein: NO: 76), wherein: X₂₅ is G, M, R, or S; X₂₆ is A, G, H, L, R,or S; X₂₇ X₂₅ is G, M, R, or S; is D, H, R, or T; X₂₈ is A, P, R, S, T,or V; X₂₉ is F, X₂₆ is A, G, H, L, R, or S; G, L, M, P, or S; X₃₀ is G,L, P, S, V, or W; and X₃₁ X₂₇ is D, H, N, R, S, T, or W; is G, P, R, orS X₂₈ is A, N, P, R, S, T, or V; X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁ (SEQ ID NO: 78),wherein: X₂₉ is F, G, L, M, P, Q, R, S, or Y; X₂₅ is G, M, R, or S; X₂₆is G, L, or S; X₂₇ is R or T; X₃₀ is I, G, L, P, S, V, or W; and X₂₈ isA, P, or S; X₂₉ is F, G, M, or S; X₃₀ is G, P, S, X₃₁ is G, P, R, or SV, or W; and X₃₁ is G, R, or S X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁ (SEQ ID NO: 79),wherein: X₂₅ is G, M, R, or S; X₂₆ is G, L, or S; X₂₇ is R; X₂₈ is A orS; X₂₉ is G or M; X₃₀ is G, P, S, or W; and X₃₁ is G, R, or SX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁ (SEQ ID NO: 80), wherein: X₂₅ is M; X₂₆ is L; X₂₇is R; X₂₈ is A or S; X₂₉ is G; X₃₀ is W; and X₃₁ is R

TABLE 9D-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 4CSubgenus of Expanded Core CM Expanded Core CM Consensus 4C Consensus 4CX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂ (SEQ ID X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂ (SEQ ID NO:82), NO: 81), wherein: wherein: X₂₅ is G, M, R, or S; X₂₆ is A, G, H, L,R, X₂₅ is G, M, R, or S; or S; X₂₇ is D, H, R, or T; X₂₈ is A, P, R, S,T, or V; X₂₆ is A, G, H, L, R, or S; X₂₉ is F, G, L, M, P, or S; X₃₀ isG, L, P, S, V, or W; X₂₇ is D, H, N, R, S, T, or W; X₃₁ is G, P, R, orS; and X₃₂ is G, L, R, S, or V X₂₈ is A, N, P, R, S, T, or V;X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂ (SEQ ID NO: 83), X₂₉ is F, G, L, M, P, Q, R, S,or Y; wherein: X₂₅ is G, M, R, or S; X₂₆ is G, L, or S; X₂₇ X₃₀ is I, G,L, P, S, V, or W; is R or T; X₂₈ is A, P, or S; X₂₉ is F, G, M, or S;X₃₀ X₃₁ is G, P, R, or S; and is G, P, S, V, or W; X₃₁ is G, R, or S;and X_(32 is G,) X₃₂ is G, L, R, S, or V L, S, or VX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂ (SEQ ID NO: 84), wherein: X₂₅ is G, M, R, or S;X₂₆ is G, L, or S; X₂₇ is R; X₂₈ is A or S; X₂₉ is G or M; X₃₀ is G, P,S, or W; X₃₁ is G, R, or S; and X₃₂ is G, L, S, or VX₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂ (SEQ ID NO: 85), wherein: X₂₅ is M; X₂₆ is L;X₂₇ is R; X₂₈ is A or S; X₂₉ is G; X₃₀ is W; X₃₁ is R; and X₃₂ is G, L,or S

TABLE 9E-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 5ASubgenus of Expanded Core CM Expanded Core CM Consensus 5A Consensus 5AX₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 86), X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 87),wherein: wherein: X₃₅ is G, I, S, or V X₃₆ is G, L, S, V, or W;X_(37 is G,) X₃₅ is A, E, G, H, I, L, N, P, S, or V; Q, R, or S; X₃₈ isR; X₃₉ is G, S, or V; and X₄₀ is A, X₃₆ is G, K, L, S, V, or W; G, L, S,or V X₃₇ is G, I, P, Q, R, or S; X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 88),wherein: X₃₈ is R; X₃₅ is G, I, S, or V X₃₆ is V; X₃₇ is S; X₃₈ is R;X₃₉ is G, K, R, S, or V; and X₃₉ is S; and X₄₀ is A or V X₄₀ is A, C, G,L, M, P, S, V, or Y X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 89), wherein: X₃₅ isI; X₃₆ is V; X₃₇ is S; X₃₈ is R; X₃₉ is S; and X₄₀ is A or V

TABLE 9E-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 5BSubgenus of Expanded Core CM Expanded Core CM Consensus 5B Consensus 5BX₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 91), NO:90), wherein: wherein: X₃₄ is A, G, K, M, P, Q, S, V, or Y; X₃₄ is A, G,K, S, V, or Y; and X₃₅ is G, I, S, or V X₃₅ is A, E, G, H, I, L, N, P,S, or V; X₃₆ is G, L, S, V, or W; X₃₇ is G, Q, R, or S; X₃₈ is X₃₆ is G,K, L, S, V, or W; R; X₃₉ is G, S, or V; and X₄₀ is A, G, L, S, or V X₃₇is G, I, P, Q, R, or S; X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 92), wherein:X₃₈ is R; X₃₄ is G, S, V, or Y; X₃₅ is G, I, S, or V X₃₆ is V; X₃₉ is G,K, R, S, or V; and X₃₇ is 5; X₃₈ is R; X₃₉ is S; and X₄₀ is A or V X₄₀is A, C, G, L, M, P, S, V, or Y X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 93),wherein: X₃₄ is Y; X₃₅ is I; X₃₆ is V; X₃₇ is 5; X₃₈ is R; X₃₉ is S; andX₄₀ is A or V

TABLE 9E-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 5CSubgenus of Expanded Core CM Expanded Core CM Consensus 5C Consensus 5CX₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO:95), NO: 94), wherein: wherein: X₃₃ is G, P, Q, S, or T; X₃₄ is A, G, K,S, X₃₃ is G, K, P, Q, S, or T; V, or Y; and X₃₅ is G, I, S, or V X₃₆ isG, L, S, V, or X₃₄ is A, G, K, M, P, Q, S, V, or Y; W; X₃₇ is G, Q, R,or S; X₃₈ is R; X₃₉ is G, S, or V; X₃₅ is A, E, G, H, I, L, N, P, S, orV; and X₄₀ is A, G, L, S, or V X₃₆ is G, K, L, S, V, or W;X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 96), X₃₇ is G, I, P, Q, R, or S;wherein: X₃₃ is G, Q, or S; X₃₄ is G, S, V, or Y; X₃₅ X₃₈ is R; is G, I,S, or V X₃₆ is V; X₃₇ is S; X₃₈ is R; X₃₉ is G, K, R, S, or V; and X₃₉is S; and X₄₀ is A or V X₄₀ is A, C, G, L, M, P, S, V, or YX₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 97), wherein: X₃₃ is G, Q, or S;X₃₄ is Y; X₃₅ is I; X₃₆ is V; X₃₇ is S; X₃₈ is R; X₃₉ is S; and X₄₀ is Aor V

TABLE 9F Matriptase Cleavable Expanded Core CM Consensus Sequence 6Subgenus of Expanded Core CM Expanded Core CM Consensus 6 Consensus 6X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 98), X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 99),wherein: wherein: X₄₁ is G, K, R, S, or T; X₄₂ is A, E, G, L, M, R, orS; X₄₁ is G, K, P, R, S, or T; X₄₃ is G, K, L, N, R, S, or V; X₄₄ is Ror Y; X₄₅ is A, X₄₂ is A, E, G, I, L, M, R, or S; F, G, P, or S; and X₄₆is F, G, M, P, R, S, or V X₄₃ is A, G, K, L, N, R, S, or V;X₄₁X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 100), wherein: X₄₄ is F, H, L, R, or Y;X₄₁ is G, R, S, or T; X₄₂ is A, E, G, M, or S; X₄₃ is X₄₅ is A, F, G, H,P, or S; and G, L, S, or V; X₄₄ is R or Y; X₄₅ is A, G, P, or S; and X₄₆is F, G, M, N, P, R, S, or V X₄₆ is F, G, M, P, R, S, or VX₄₁X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 101), wherein: X₄₁ is G, R, or S; X₄₂ isA, G, or S; X₄₃ is L, S, or V; X₄₄ is R; X₄₅ is A; and X₄₆ is M or PX₄₁X₄₂X₄₃X₄₄X₄₅X₄₆ (SEQ ID NO: 102), wherein: X₄₁ is G, R, or S; X₄₂ isA; X₄₃ is L, S or V; X₄₄ is R; X₄₅ is A; and X₄₆ is M or P

TABLE 9G-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 7ASubgenus of Expanded Core CM Expanded Core CM Consensus 7A Consensus 7AX₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 103), X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO:104), wherein: wherein: X₅₀ is E, P, S, V, or W; X₅₁ is A, P, R, S, V,or Y; X₅₀ is A, E, K, L, P, S, T, V, W, or Y; X₅₂ is E, G, H, L, P, orV; X₅₃ is G, K, L, or R; X₅₄ X₅₁ is A, I, L, P, R, S, V, or Y; is Q orR; and X₅₅ is A, G, H, M, R, or S X₅₂ is E, G, H, L, P, or V;X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 105), wherein: X₅₃ is G, K, L, or R; X₅₀is P or V; X₅₁ is A, P, or R; X₅₂ is E, G, P, or V; X₅₄ is Q or R; andand X₅₃ is G or R; X₅₄ is R; X₅₅ is G, M, or S X₅₅ is A, G, H, M, R, orS X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 106), wherein: X₅₀ is P or V; X₅₁ is Aor R; X₅₂ is G or V; X₅₃ is G or R; X₅₄ is R; and X₅₅ is M or SX₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 107), wherein: X₅₀ is P or V; X₅₁ is A;X₅₂ is G or V; X₅₃ is R; X₅₄ is R; and X₅₅ is M or S

TABLE 9G-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 7BSubgenus of Expanded Core CM Expanded Core CM Consensus 7B Consensus 7BX₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 109),NO: 108), wherein: wherein: X₄₉ is G, K, P, Q, S, or V; X₅₀ is E, P, S,X₄₉ is E, G, K, P, Q, S, T, or V; V, or W; X₅₁ is A, P, R, S, V, or Y;X₅₂ is E, G, H, X₅₀ is A, E, K, L, P, S, T, V, W, or Y; L, P, or V; X₅₃is G, K, L, or R; X₅₄ is Q or R; and X₅₁ is A, I, L, P, R, S, V, or Y;X₅₅ is A, G, H, M, R, or S X₅₂ is E, G, H, L, P, or V;X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO:: 110), X₅₃ is G, K, L, or R; wherein:X₄₉ is G, P, S, or V; X₅₀ is P or V; X₅₁ is X₅₄ is Q or R; and A, P, orR; X₅₂ is E, G, P, or V; X₅₃ is G or R; X₅₄ is X₅₅ is A, G, H, M, R, orS R; and X₅₅ is G, M, or S X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQ ID NO: 111),wherein: X₄₉ is G, P, S, or V; X₅₀ is P or V; X₅₁ is A or R; X₅₂ is G orV; X₅₃ is G or R; X₅₄ is R; and X₅₅ is M or S X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅ (SEQID NO: 112), wherein: X₄₉ is G, S, or V; X₅₀ is P or V; X₅₁ is A; X₅₂ isG or V; X₅₃ is R; X₅₄ is R; and X₅₅ is M or S

TABLE 9G-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 7CSubgenus of Expanded Core CM Expanded Core CM Consensus 7C Consensus 7CX₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆ (SEQ ID X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆ (SEQ ID NO:114), NO: 113), wherein: wherein: X₄₉ is G, K, P, Q, S, or V; X₅₀ is E,P, S, X₄₉ is E, G, K, P, Q, S, T, or V; V, or W; X₅₁ is A, P, R, S, V,or Y; X₅₂ is E, G, H, X₅₀ is A, E, K, L, P, S, T, V, W, or Y; L, P, orV; X₅₃ is G, K, L, or R; X₅₄ is Q or R; X₅₅ is X₅₁ is A, I, L, P, R, S,V, or Y; A, G, H, M, R, or S; and X₅₆ is G, L, M, P, S, or W X₅₂ is E,G, H, L, P, or V; X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆ (SEQ ID NO: 115), X₅₃ is G,K, L, or R; wherein: X₄₉ is G, P, S, or V; X₅₀ is P or V; X₅₁ is X₅₄ isQ or R; A, P, or R; X₅₂ is E, G, P, or V; X₅₃ is G or R; X₅₄ is X₅₅ isA, G, H, M, R, or S; and R; X₅₅ is G, M, or S; and X₅₆ is G, L, M, P, S,or W X₅₆ is F, G, L, M, P, S, or W X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆ (SEQ ID NO:116), wherein: X₄₉ is G, P, S, or V; X₅₀ is P or V; X₅₁ is A or R; X₅₂is G or V; X₅₃ is G or R; X₅₄ is R; X₅₅ is M or S; and X₅₆ is G, L, P,S, or W X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆ (SEQ ID NO: 117), wherein: X₄₉ is G, S,or V; X₅₀ is P or V; X₅₁ is A; X₅₂ is G or V; X₅₃ is R; X₅₄ is R; X₅₅ isM or S; and X₅₆ is G, L, or S

TABLE 9H-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 8ASubgenus of Expanded Core CM Expanded Core CM Consensus 8A Consensus 8AX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂ (SEQ ID NO: 118), X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂ (SEQ ID NO:119), wherein: wherein: X₅₇ is A, G, I, K, S, or T; X₅₈ is R; X₅₉ is 5;X₆₀ is X₅₇ is A, G, I, K, P, S, or T; F, M, or R; X₆₁ is A, I, L, R, orW; and X₆₂ is G, L, X₅₈ is R or T; M, P, Q, R, S, or V X₅₉ is H, M, orS; X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂ (SEQ ID NO: 120), wherein: X₆₀ is F, M, or R; X₅₇is G or K; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A, X₆₁ is A, G, I, L, P,Q, R, S, V, or W; L, R, or W; and X₆₂ is G, L, M, P, R, or S andX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂ (SEQ ID NO: 121), wherein: X₆₂ is A, G, L, M, P, Q,R, S, T, V, or W X₅₇ is G; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A or L;and X₆₂ is G, L, M, R, or S X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂ (SEQ ID NO: 122),wherein: X₅₇ is G; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A or L; and X₆₂is L or M

TABLE 9H-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 8BSubgenus of Expanded Core CM Expanded Core CM Consensus 8B Consensus 8BX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃ (SEQ ID X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃ (SEQ ID NO: 124),NO: 123), wherein: wherein: X₅₇ is A, G, I, K, S, or T; X₅₈ is R; X₅₉ isX₅₇ is A, G, I, K, P, S, or T; S; X₆₀ is F, M, or R; X₆₁ is A, I, L, R,or W; X₆₂ is X₅₈ is R or T; G, L, M, P, Q, R, S, or V; and X₆₃ is A, G,P, R, S, X₅₉ is H, M, or S; W, or Y X₆₀ is F, M, or R;X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃ (SEQ ID NO: 125), X₆₁ is A, G, I, L, P, Q, R, S,V, or W; wherein: X₅₇ is G or K; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₂ is A,G, L, M, P, Q, R, S, T, V, or X₆₁ is A, L, R, or W; X₆₂ is G, L, M, P,R, or S; and W; and X₆₃ is A, G, P, R, S, or W X₆₃ is A, G, K, M, P, R,S, W, or Y X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃ (SEQ ID NO: 126), wherein: X₅₇ is G;X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A or L; X₆₂ is G, L, M, R, or S;and X₆₃ is A, G, P, R, or S X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃ (SEQ ID NO: 127),wherein: X₅₇ is G; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A or L; X₆₂ is Lor M; and X₆₃ is G, P, or S

TABLE 9H-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 8CSubgenus of Expanded Core CM Expanded Core CM Consensus 8C Consensus 8CX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄ (SEQ ID X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄ (SEQ ID NO:129), NO: 128), wherein: wherein: X₅₇ is A, G, I, K, S, or T; X₅₈ is R;X₅₉ is X₅₇ is A, G, I, K, P, S, or T; S; X₆₀ is F, M, or R; X₆₁ is A, I,L, R, or W; X₆₂ is X₅₈ is R or T; G, L, M, P, Q, R, S, or V; X₆₃ is A,G, P, R, S, W, or X₅₉ is H, M, or S; Y; and X₆₄ is F, G, L, P, or S X₆₀is F, M, or R; X₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄ (SEQ ID NO: 130), X₆₁ is A, G,I, L, P, Q, R, S, V, or W; wherein: X₅₇ is G or K; X₅₈ is R; X₅₉ is S;X₆₀ is M; X₆₂ is A, G, L, M, P, Q, R, S, T, V, or X₆₁ is A, L, R, or W;X₆₂ is G, L, M, P, R, or S; X₆₃ W; is A, G, P, R, S, or W; and X₆₄ is F,G, L, P, or S X₆₃ is A, G, K, M, P, R, S, W, or Y; andX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄ (SEQ ID NO: 131), X₆₄ is F, G, I, L, P, Q, S,or Y wherein: X₅₇ is G; X₅₈ is R; X₅₉ is S; X₆₀ is M; X₆₁ is A or L; X₆₂is G, L, M, R, or S; X₆₃ is A, G, P, R, or S; and X₆₄ is F, G, P, or SX₅₇X₅₈X₅₉X₆₀X₆₁X₆₂X₆₃X₆₄ (SEQ ID NO: 132), wherein: X₅₇ is G; X₅₈ is R;X₅₉ is S; X₆₀ is M; X₆₁ is A or L; X₆₂ is L or M; X₆₃ is G, P, or S; andX₆₄ is G, P, or S

TABLE 9I-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 9ASubgenus of Expanded Core CM Expanded Core CM Consensus 9A Consensus 9AX₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 133), X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO:134), wherein: wherein: X₆₇ is I or L; X₆₈ is A, G, P, or V; X₆₉ is R;X₇₀ is A, X₆₇ is I, L, or S; M, P, R, S, or T; X₇₁ is G, K, L, R, S, orV; and X₇₂ X₆₈ is A, G, K, P, R, or V; is F, G, I, L, M, S, or V X₆₉ isL, R or S; X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 135), wherein: X₇₀ is A, K, M,P, R, S, or T; X₆₇ is L; X₆₈ is A, G, P, or V; X₆₉ is R; X₇₀ is A, M,X₇₁ is F, G, H, I, K, L, M, P, R, S, or V; P, R, or S; X₇₁ is G, K, L,S, or V; and X₇₂ is F, G, I, and L, S, or V X₇₂ is F, G, I, L, M, P, R,S, T, V, W, or Y X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 136), wherein: X₆₇ is L;X₆₈ is A, G, or P; X₆₉ is R; X₇₀ is A or S; X₇₁ is G or L; and X₇₂ is Ior L X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 137), wherein: X₆₇ is L; X₆₈ is A orP; X₆₉ is R; X₇₀ is A; X₇₁ is G or L; and X₇₂ is I or L

TABLE 9I-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 9BSubgenus of Expanded Core CM Expanded Core CM Consensus 9B Consensus 9BX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 139),NO: 138), wherein: wherein: X₆₆ is G, P, R, S, or T; X₆₇ is I or L; X₆₈is X₆₆ is G, K, P, Q, R, S, or T; A, G, P, or V; X₆₉ is R; X₇₀ is A, M,P, R, S, or T; X₆₇ is I, L, or S; X₇₁ is G, K, L, R, S, or V; and X₇₂ isF, G, I, L, M, X₆₈ is A, G, K, P, R, or V; S, or V X₆₉ is L, R or S;X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 140), X₇₀ is A, K, M, P, R, S, or T;wherein: X₆₆ is G, P, R, or S; X₆₇ is L; X₆₈ is A, G, X₇₁ is F, G, H, I,K, L, M, P, R, S, or P, or V; X₆₉ is R; X₇₀ is A, M, P, R, or S; X₇₁ isG, V; and K, L, S, or V; and X₇₂ is F, G, I, L, S, or V X₇₂ is F, G, I,L, M, P, R, S, T, V, W, X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 141), or Ywherein: X₆₆ is P; X₆₇ is L; X₆₈ is A, G, or P; X₆₉ is R; X₇₀ is A or S;X₇₁ is G or L; and X₇₂ is I or L X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 142),wherein: X₆₆ is P; X₆₇ is L; X₆₈ is A or P; X₆₉ is R; X₇₀ is A; X₇₁ is Gor L; and X₇₂ is I or L

TABLE 9I-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 9CSubgenus of Expanded Core CM Expanded Core CM Consensus 9C Consensus 9CX₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO:144), NO: 143), wherein: wherein: X₆₅ is G, K, P, R, S, or V; X₆₆ is G,P, R, X₆₅ is A, G, I, K, P, R, S, or V; S, or T; X₆₇ is I or L; X₆₈ isA, G, P, or V; X₆₉ is R; X₆₆ is G, K, P, Q, R, S, or T; X₇₀ is A, M, P,R, S, or T; X₇₁ is G, K, L, R, S, or V; X₆₇ is I, L, or S; and X₇₂ is F,G, I, L, M, S, or V X₆₈ is A, G, K, P, R, or V; X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂(SEQ ID NO: 145), X₆₉ is L, R or S; wherein: X₆₅ is G, P, R, or S; X₆₆is G, P, R, or S; X₇₀ is A, K, M, P, R, S, or T; X₆₇ is L; X₆₈ is A, G,P, or V; X₆₉ is R; X₇₀ is A, M, X₇₁ is F, G, H, I, K, L, M, P, R, S, orP, R, or S; X₇₁ is G, K, L, S, or V; and X₇₂ is F, G, V; and I, L, S, orV X₇₂ is F, G, I, L, M, P, R, S, T, V, W, X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQID NO: 146), or Y wherein: X₆₅ is G, P, R, or S; X₆₆ is P; X₆₇ is L; X₆₈is A, G, or P; X₆₉ is R; X₇₀ is A or S; X₇₁ is G or L; and X₇₂ is I or LX₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂ (SEQ ID NO: 147), wherein: X₆₅ is R; X₆₆ is P;X₆₇ is L; X₆₈ is A or P; X₆₉ is R; X₇₀ is A; X₇₁ is G or L; and X₇₂ is Ior L

TABLE 9J-1 Matriptase Cleavable Expanded Core CM Consensus Sequence 10ASubgenus of Expanded Core CM Expanded Core CM Consensus 10A Consensus10A X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO:149), NO: 148), wherein: wherein: X₇₃ is G, N, P, S, T, or V; X₇₄ is E,L, T or X₇₃ is G, H, N, P, R, S, T, or V; V; X₇₅ is R or S; X₇₆ is K orR; X₇₇ is M, R, or S; X₇₄ is E, L, Q, S, T or V; X₇₈ is G, L, M, S, orW; and X₇₉ is A, G, I, M, P, S, X₇₅ is L, R, or S; or V X₇₆ is H, K, orR; X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 150), X₇₇ is A, M, R, or S;wherein: X₇₃ is G, N, P, S, or V; X₇₄ is E, T or V; X₇₈ is G, L, M, R,S, or W; and X₇₅ is R or S; X₇₆ is K or R; X₇₇ is M or R; X₇₈ is G, X₇₉is A, G, I, M, N, P, S, V, or Y L, M, S, or W; and X₇₉ is A, G, I, M, P,S, or V X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO: 151), wherein: X₇₃ is G, P, S,or V; X₇₄ is E or V; X₇₅ is S; X₇₆ is K or R; X₇₇ is R; X₇₈ is L, S, orW; and X₇₉ is G, I, M, P, S, or V X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉ (SEQ ID NO:152), wherein: X₇₃ is G, P, or S; X₇₄ is E; X₇₅ is S; X₇₆ is K or R; X₇₇is R; X₇₈ is L or W; and X₇₉ is M

TABLE 9J-2 Matriptase Cleavable Expanded Core CM Consensus Sequence 10BSubgenus of Expanded Core CM Expanded Core CM Consensus 10B Consensus10B X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO:154), NO: 153), wherein: wherein: X₇₄ is E, L, T or V; X₇₅ is R or S;X₇₆ is K X₇₄ is E, L, Q, S, T or V; or R; X₇₇ is M, R, or S; X₇₈ is G,L, M, S, or W; X₇₉ X₇₅ is L, R, or S; is A, G, I, M, P, S, or V; and X₈₀is G, I, L, N, P, S, X₇₆ is H, K, or R; or V X₇₇ is A, M, R, or S;X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO: 155), X₇₈ is G, L, M, R, S, or W;wherein: X₇₄ is E, T or V; X₇₅ is R or S; X₇₆ is K or X₇₉ is A, G, I, M,N, P, S, V, or Y; R; X₇₇ is M or R; X₇₈ is G, L, M, S, or W; X₇₉ is A,and G, I, M, P, S, or V; and X₈₀ is G, I, L, N, P, S, or V X₈₀ is G, I,L, N, P, S, V, or W X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO: 156), wherein: X₇₄is E or V; X₇₅ is S; X₇₆ is K or R; X₇₇ is R; X₇₈ is L, S, or W; X₇₉ isG, I, M, P, S, or V; and X₈₀ is G, L, N, P, S, or VX₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO: 157), wherein: X₇₄ is E; X₇₅ is S; X₇₆is K or R; X₇₇ is R; X₇₈ is L or W; X₇₉ is M; and X₈₀ is G, P, or S

TABLE 9J-3 Matriptase Cleavable Expanded Core CM Consensus Sequence 10CSubgenus of Expanded Core CM Expanded Core CM Consensus 10C Consensus10C X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ IDNO: 159), NO: 158), wherein: wherein: X₇₃ is G, N, P, S, T, or V; X₇₄ isE, L, T or X₇₃ is G, H, N, P, R, S, T, or V; V; X₇₅ is R or S; X₇₆ is Kor R; X₇₇ is M, R, or S; X₇₄ is E, L, Q, S, T or V; X₇₈ is G, L, M, S,or W; X₇₉ is A, G, I, M, P, S, or X₇₅ is L, R, or S; V; and X₈₀ is G, I,L, N, P, S, or V X₇₆ is H, K, or R; X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO:160), X₇₇ is A, M, R, or S; wherein: X₇₃ is G, N, P, S, or V; X₇₄ is E,T or V; X₇₈ is G, L, M, R, S, or W; X₇₅ is R or S; X₇₆ is K or R; X₇₇ isM or R; X₇₈ is G, X₇₉ is A, G, I, M, N, P, S, V, or Y; L, M, S, or W;X₇₉ is A, G, I, M, P, S, or V; and X₈₀ and is G, I, L, N, P, S, or V X₈₀is G, I, L, N, P, S, V, or W X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO: 161),wherein: X₇₃ is G, P, S, or V; X₇₄ is E or V; X₇₅ is S; X₇₆ is K or R;X₇₇ is R; X₇₈ is L, S, or W; X₇₉ is G, I, M, P, S, or V; and X₈₀ is G,L, N, P, S, or V X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ (SEQ ID NO: 162), wherein: X₇₃is G, P, or S; X₇₄ is E; X₇₅ is S; X₇₆ is K or R; X₇₇ is R; X₇₈ is L orW; X₇₉ is M; and X₈₀ is G, P, or S

In some embodiments, the CM comprises a core CM consensus 1 sequencecomprising the amino acid sequence AAPRS (SEQ ID NO: 163). In someembodiments, the CM comprises an expanded core CM consensus 1 sequencecomprising the amino acid sequence AAPRSF (SEQ ID NO: 164).

In some embodiments, the CM comprises a core CM consensus 2 sequencecomprising the amino acid sequence SRRVP (SEQ ID NO: 165). In someembodiments, the CM comprises an expanded core CM consensus 2 sequencecomprising an amino acid sequence selected from the group consisting ofQSRRVP (SEQ ID NO: 166), QTRRVP (SEQ ID NO: 167), SRRVPL (SEQ ID NO:168), SRRVPV (SEQ ID NO: 169), QSRRVPL (SEQ ID NO: 170), QSRRVPV (SEQ IDNO: 171), QTRRVPL (SEQ ID NO: 172), and QTRRVPV (SEQ ID NO: 173).

In some embodiments, the CM comprises the amino acid sequence QSRRVP(SEQ ID NO: 166). In some embodiments, the CM comprises the amino acidsequence QTRRVP (SEQ ID NO: 167). In some embodiments, the CM comprisesthe amino acid sequence SRRVPL (SEQ ID NO: 168). In some embodiments,the CM comprises the amino acid sequence SRRVPV (SEQ ID NO: 169). Insome embodiments, the CM comprises the amino acid sequence QSRRVPL (SEQID NO: 170). In some embodiments, the CM comprises the amino acidsequence QSRRVPV (SEQ ID NO: 171). In some embodiments, the CM comprisesthe amino acid sequence QTRRVPL (SEQ ID NO: 172). In some embodiments,the CM comprises the amino acid sequence QTRRVPV (SEQ ID NO: 173).

In some embodiments, the CM comprises a core CM consensus 3 sequencecomprising the amino acid sequence PPLGR (SEQ ID NO: 174). In someembodiments, the CM comprises an expanded core CM consensus 3 sequencecomprising an amino acid sequence selected from the group consisting ofGPPLGR (SEQ ID NO: 175), SPPLGR (SEQ ID NO: 176), CGPPLGR (SEQ ID NO:177), CSPPLGR (SEQ ID NO: 178), GGPPLGR (SEQ ID NO: 179), GSPPLGR (SEQID NO: 180), SGPPLGR (SEQ ID NO: 181), SSPPLGR (SEQ ID NO: 182),GCGPPLGR (SEQ ID NO: 183), GCSPPLGR (SEQ ID NO: 184), GGGPPLGR (SEQ IDNO: 185), GGSPPLGR (SEQ ID NO: 186), GSGPPLGR (SEQ ID NO: 187), GSSPPLGR(SEQ ID NO: 188), SCGPPLGR (SEQ ID NO: 189), SCSPPLGR (SEQ ID NO: 190),SGGPPLGR (SEQ ID NO: 191), SGSPPLGR (SEQ ID NO: 192), SSGPPLGR (SEQ IDNO: 193), and SSSPPLGR (SEQ ID NO: 194).

In some embodiments, the CM comprises the amino acid sequence GPPLGR(SEQ ID NO: 175). In some embodiments, the CM comprises the amino acidsequence SPPLGR (SEQ ID NO: 176). In some embodiments, the CM comprisesthe amino acid sequence CGPPLGR (SEQ ID NO: 177). In some embodiments,the CM comprises the amino acid sequence CSPPLGR (SEQ ID NO: 178). Insome embodiments, the CM comprises the amino acid sequence GGPPLGR (SEQID NO: 179). In some embodiments, the CM comprises the amino acidsequence GSPPLGR (SEQ ID NO: 180). In some embodiments, the CM comprisesthe amino acid sequence SGPPLGR (SEQ ID NO: 181). In some embodiments,the CM comprises the amino acid sequence SSPPLGR (SEQ ID NO: 182). Insome embodiments, the CM comprises the amino acid sequence GCGPPLGR (SEQID NO: 183). In some embodiments, the CM comprises the amino acidsequence GCSPPLGR (SEQ ID NO: 184). In some embodiments, the CMcomprises the amino acid sequence GGGPPLGR (SEQ ID NO: 185). In someembodiments, the CM comprises the amino acid sequence GGSPPLGR (SEQ IDNO: 186). In some embodiments, the CM comprises the amino acid sequenceGSGPPLGR (SEQ ID NO: 187). In some embodiments, the CM comprises theamino acid sequence GSSPPLGR (SEQ ID NO: 188). In some embodiments, theCM comprises the amino acid sequence SCGPPLGR (SEQ ID NO: 189). In someembodiments, the CM comprises the amino acid sequence SCSPPLGR (SEQ IDNO: 190). In some embodiments, the CM comprises the amino acid sequenceSGGPPLGR (SEQ ID NO: 191). In some embodiments, the CM comprises theamino acid sequence SGSPPLGR (SEQ ID NO: 192). In some embodiments, theCM comprises the amino acid sequence SSGPPLGR (SEQ ID NO: 193). In someembodiments, the CM comprises the amino acid sequence SSSPPLGR (SEQ IDNO: 194).

In some embodiments, the CM comprises a core CM consensus 4 sequencecomprising the amino acid sequence LRSGW (SEQ ID NO: 195). In someembodiments, the CM comprises an expanded core CM consensus 4 sequencecomprising an amino acid sequence selected from the group consisting ofMLRSGW (SEQ ID NO: 196), MLRSGWR (SEQ ID NO: 197), MLRSGWRG (SEQ ID NO:198), MLRSGWRL (SEQ ID NO: 199), and MLRSGWRS (SEQ ID NO: 200).

In some embodiments, the CM comprises the amino acid sequence MLRSGW,(SEQ ID NO: 196). In some embodiments, the CM comprises the amino acidsequence MLRSGWR (SEQ ID NO: 197). In some embodiments, the CM comprisesthe amino acid sequence MLRSGWRG (SEQ ID NO: 198). In some embodiments,the CM comprises the amino acid sequence MLRSGWRL (SEQ ID NO: 199). Insome embodiments, the CM comprises the amino acid sequence MLRSGWRS (SEQID NO: 200).

In some embodiments, the CM comprises a core CM consensus 5 sequencecomprising the amino acid sequence VSRSA (SEQ ID NO: 201). In someembodiments, the CM comprises an expanded core CM consensus 5 sequencecomprising an amino acid sequence selected from the group consisting ofIVSRSA (SEQ ID NO: 202), YIVSRSA (SEQ ID NO: 203), and QYIVSRSA (SEQ IDNO: 204).

In some embodiments, the CM comprises the amino acid sequence IVSRSA(SEQ ID NO: 202). In some embodiments, the CM comprises the amino acidsequence YIVSRSA (SEQ ID NO: 203). In some embodiments, the CM comprisesthe amino acid sequence QYIVSRSA (SEQ ID NO: 204).

In some embodiments, the CM comprises a core CM consensus 6 sequencecomprising the amino acid sequence ALRAP (SEQ ID NO: 205). In someembodiments, the CM comprises an expanded core CM consensus 6 sequencecomprising the amino acid sequence RALRAP (SEQ ID NO: 206).

In some embodiments, the CM comprises a core CM consensus 7 sequencecomprising the amino acid sequence PAGRR (SEQ ID NO: 207). In someembodiments, the CM comprises an expanded core CM consensus 7 sequencecomprising an amino acid sequence selected from the group consisting ofPAGRRS (SEQ ID NO: 208), PAGRRSL (SEQ ID NO: 209), VPAGRRS (SEQ ID NO:210), and VPAGRRSL (SEQ ID NO: 211).

In some embodiments, the CM comprises the amino acid sequence PAGRRS(SEQ ID NO: 208). In some embodiments, the CM comprises the amino acidsequence PAGRRSL (SEQ ID NO: 209). In some embodiments, the CM comprisesthe amino acid sequence VPAGRRS (SEQ ID NO: 210). In some embodiments,the CM comprises the amino acid sequence VPAGRRSL (SEQ ID NO: 211).

In some embodiments, the CM comprises a core CM consensus 8 sequencecomprising the amino acid sequence GRSML (SEQ ID NO: 212). In someembodiments, the CM comprises an expanded core CM consensus 8 sequencecomprising an amino acid sequence selected from the group consisting ofGRSMLL (SEQ ID NO: 213), GRSMLM (SEQ ID NO: 214), GRSMLLG (SEQ ID NO:215), GRSMLLP (SEQ ID NO: 216), GRSMLLS (SEQ ID NO: 217), GRSMLMG (SEQID NO: 218), GRSMLMP (SEQ ID NO: 219), GRSMLMS (SEQ ID NO: 220),GRSMLLGG (SEQ ID NO: 221), GRSMLLPG (SEQ ID NO: 222), GRSMLLSG (SEQ IDNO: 223), GRSMLMGG (SEQ ID NO: 224), GRSMLMPG (SEQ ID NO: 225), GRSMLMSG(SEQ ID NO: 226), GRSMLLGP (SEQ ID NO: 227), GRSMLLPP (SEQ ID NO: 228),GRSMLLSP (SEQ ID NO: 229), GRSMLMGP (SEQ ID NO: 230), GRSMLLPP (SEQ IDNO: 231), GRSMLMSP (SEQ ID NO: 232), GRSMLLGS (SEQ ID NO: 233), GRSMLLPS(SEQ ID NO: 234), GRSMLLSS (SEQ ID NO: 235), GRSMLMGS (SEQ ID NO: 236),GRSMLMPS (SEQ ID NO: 237), and GRSMLMSS (SEQ ID NO: 238).

In some embodiments, the CM comprises the amino acid sequence GRSMLL(SEQ ID NO: 213). In some embodiments, the CM comprises the amino acidsequence GRSMLM (SEQ ID NO: 214). In some embodiments, the CM comprisesthe amino acid sequence GRSMLLG (SEQ ID NO: 215). In some embodiments,the CM comprises the amino acid sequence GRSMLLP (SEQ ID NO: 216). Insome embodiments, the CM comprises the amino acid sequence GRSMLLS (SEQID NO: 217). In some embodiments, the CM comprises the amino acidsequence GRSMLMG (SEQ ID NO: 218). In some embodiments, the CM comprisesthe amino acid sequence GRSMLMP (SEQ ID NO: 219). In some embodiments,the CM comprises the amino acid sequence GRSMLMS (SEQ ID NO: 220). Insome embodiments, the CM comprises the amino acid sequence GRSMLLGG (SEQID NO: 221). In some embodiments, the CM comprises the amino acidsequence GRSMLLPG (SEQ ID NO: 222). In some embodiments, the CMcomprises the amino acid sequence GRSMLLSG (SEQ ID NO: 223). In someembodiments, the CM comprises the amino acid sequence GRSMLMGG (SEQ IDNO: 224). In some embodiments, the CM comprises the amino acid sequenceGRSMLMPG (SEQ ID NO: 225). In some embodiments, the CM comprises theamino acid sequence GRSMLMSG (SEQ ID NO: 226). In some embodiments, theCM comprises the amino acid sequence GRSMLLGP (SEQ ID NO: 227). In someembodiments, the CM comprises the amino acid sequence GRSMLLPP (SEQ IDNO: 228). In some embodiments, the CM comprises the amino acid sequenceGRSMLLSP (SEQ ID NO: 229). In some embodiments, the CM comprises theamino acid sequence GRSMLMGP (SEQ ID NO: 230). In some embodiments, theCM comprises the amino acid sequence GRSMLMPP (SEQ ID NO: 231). In someembodiments, the CM comprises the amino acid sequence GRSMLMSP (SEQ IDNO: 232). In some embodiments, the CM comprises the amino acid sequenceGRSMLLGS (SEQ ID NO: 233). In some embodiments, the CM comprises theamino acid sequence GRSMLLPS (SEQ ID NO: 234). In some embodiments, theCM comprises the amino acid sequence GRSMLLSS (SEQ ID NO: 235). In someembodiments, the CM comprises the amino acid sequence GRSMLMGS (SEQ IDNO: 236). In some embodiments, the CM comprises the amino acid sequenceGRSMLMPS (SEQ ID NO: 237). In some embodiments, the CM comprises theamino acid sequence GRSMLMSS (SEQ ID NO: 238).

In some embodiments, the CM comprises a core CM consensus 9 sequencecomprising the amino acid sequence LARAG (SEQ ID NO: 239). In someembodiments, the CM comprises an expanded core CM consensus 9 sequencecomprising an amino acid sequence selected from the group consisting ofLARAGI (SEQ ID NO: 240), LARAGL (SEQ ID NO: 241), PLARAGI (SEQ ID NO:242), PLARAGL (SEQ ID NO: 243), RPLARAGI (SEQ ID NO: 244), and RPLARAGL(SEQ ID NO: 245).

In some embodiments, the CM comprises the amino acid sequence LARAGI(SEQ ID NO: 240). In some embodiments, the CM comprises the amino acidsequence LARAGL (SEQ ID NO: 241). In some embodiments, the CM comprisesthe amino acid sequence PLARAGI (SEQ ID NO: 242). In some embodiments,the CM comprises the amino acid sequence PLARAGL (SEQ ID NO: 243). Insome embodiments, the CM comprises the amino acid sequence RPLARAGI (SEQID NO: 244). In some embodiments, the CM comprises the amino acidsequence RPLARAGL (SEQ ID NO: 245).

In some embodiments, the CM comprises a core CM consensus 10 sequencecomprising the amino acid sequence ESRRW (SEQ ID NO: 246). In someembodiments, the CM comprises an expanded core CM consensus 10 sequencecomprising an amino acid sequence selected from the group consisting ofESRRWM (SEQ ID NO: 247), ESRRWMP (SEQ ID NO: 248), and PESRRWMP (SEQ IDNO: 249).

In some embodiments, the CM comprises the amino acid sequence ESRRWM(SEQ ID NO: 247). In some embodiments, the CM comprises the amino acidsequence ESRRWMP (SEQ ID NO: 248). In some embodiments, the CM comprisesthe amino acid sequence PESRRWMP (SEQ ID NO: 249).

In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of ILPRSPAF (SEQ ID NO: 250), VAGRSMRP (SEQ IDNO: 251), VVPEGRRS (SEQ ID NO: 252), QGRAITFI (SEQ ID NO: 253), VLSKQMSF(SEQ ID NO: 254), LKGRSYYY (SEQ ID NO: 255), KRMPVQFL (SEQ ID NO: 256),PQHRIVSF (SEQ ID NO: 257), YKKFVGSL (SEQ ID NO: 258), HMMQYARH (SEQ IDNO: 259), IPFSWSRF (SEQ ID NO: 260), LSQARWRK (SEQ ID NO: 261), DISHWRRS(SEQ ID NO: 262), RKTVQHWW (SEQ ID NO: 263), RFYRNQFF (SEQ ID NO: 264),RSLVFAPI (SEQ ID NO: 265), RSPSRLKC (SEQ ID NO: 266), and RKMPNITV (SEQID NO: 267).

In some embodiments, the CM comprises the amino acid sequence ILPRSPAF(SEQ ID NO: 250). In some embodiments, the CM comprises the amino acidsequence VAGRSMRP (SEQ ID NO: 251). In some embodiments, the CMcomprises the amino acid sequence VVPEGRRS (SEQ ID NO: 252). In someembodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ IDNO: 253). In some embodiments, the CM comprises the amino acid sequenceVLSKQMSF (SEQ ID NO: 254). In some embodiments, the CM comprises theamino acid sequence LKGRSYYY (SEQ ID NO: 255). In some embodiments, theCM comprises the amino acid sequence KRMPVQFL (SEQ ID NO: 256). In someembodiments, the CM comprises the amino acid sequence PQHRIVSF (SEQ IDNO: 257). In some embodiments, the CM comprises the amino acid sequenceYKKFVGSL (SEQ ID NO: 258). In some embodiments, the CM comprises theamino acid sequence HMMQYARH (SEQ ID NO: 259). In some embodiments, theCM comprises the amino acid sequence IPFSWSRF (SEQ ID NO: 260). In someembodiments, the CM comprises the amino acid sequence LSQARWRK (SEQ IDNO: 261). In some embodiments, the CM comprises the amino acid sequenceDISHWRRS (SEQ ID NO: 262). In some embodiments, the CM comprises theamino acid sequence RKTVQHWW (SEQ ID NO: 263_). In some embodiments, theCM comprises the amino acid sequence RFYRNQFF (SEQ ID NO: 264). In someembodiments, the CM comprises the amino acid sequence RSLVFAPI (SEQ IDNO: 265). In some embodiments, the CM comprises the amino acid sequenceRSPSRLKC (SEQ ID NO: 266). In some embodiments, the CM comprises theamino acid sequence RKMPNITV (SEQ ID NO: 267).

In some embodiments, the CM includes a motif sequence that is asubstrate for at least uPA and/or matriptase and includes a core CMconsensus sequence shown in Tables 10A-10J below. In some embodiments,the motif sequence includes a subgenus, i.e., a subset, of the core CMconsensus sequence shown in Tables 10A-10D below.

TABLE 10A uPA and/or Matriptase Cleavable Core CM Consensus Sequence 11Core CM Consensus 11 Subgenus of Core CM Consensus 11X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ ID X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ ID NO:269), NO: 268), wherein: wherein: X₈₇ is D, L, S, or V; X₈₈ is C, G, N,R, S, or T; X₈₇ is D, I, L, R, S, or V; X₈₉ is D, G, or S; X₉₀ is R; X₉₁is F or S; X₉₂ is A, G, I, X₈₈ is C, G, H, I, K, N, R, S, L, T, or V;X₉₃ is H, I, N, or S; and X₉₄ is H, M, R, or Y T, or Y;X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ ID NO: 270), X₈₉ is D, G, or S; wherein:X₈₇ is L or V; X₈₈ is G, H, K, N, S, or T; X₈₉ X₉₀ is R; is G; X₉₀ is R;X₉₁ is S; X₉₂ is A or D; X₉₃ is N or R; X₉₁ is F or S; and X₉₄ is H or YX₉₂ is A, D, G, H, I, L, T, or V; X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ ID NO:271), X₉₃ is H, I, N, R, S, or T; and wherein: X₈₇ is L or V; X₈₈ is G,H, N or S; X₈₉ is G; X₉₄ is H, L, M, R, V or Y X₉₀ is R; X₉₁ is S; X₉₂is A or D; X₉₃ is N; and X₉₄ is H X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ ID NO:272), wherein: X₈₇ is L or V; X₈₈ is S; X₈₉ is G; X₉₀ is R; X₉₁ is S;X₉₂ is A or D; X₉₃ is N; and X₉₄ is H X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ (SEQ IDNO: 273), wherein: X₈₇ is L or V; X₈₈ is G or S; X₈₉ is G; X₉₀ is R; X₉₁is S; X₉₂ is A; X₉₃ is N; and X₉₄ is H

TABLE 10B uPA and/or Matriptase Cleavable Core CM Consensus Sequence 12Core CM Consensus 12 Subgenus of Core CM Consensus 12X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ (SEQ IDNO: 275), (SEQ ID NO: 274), wherein: wherein: X₉₉ is L; X₁₀₀ is N, S, orT; X₁₀₁ is G; X₁₀₂ is X₉₉ is D, I, L, R, S, or V; R; X₁₀₃ is S; X₁₀₄ isA, D, or H; X₁₀₅ is N or R; and X₁₀₀ is C, G, H, I, K, N, R, S, T X₁₀₆is H, L, V, or Y or Y; X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ (SEQ ID NO: 276),X₁₀₁ is D, G, or S; wherein: X₉₉ is L or V; X₁₀₀ is G, H, K, N, S, or T;X₁₀₁ X₁₀₂ is R; is G; X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A or D; X₁₀₅ is N orX₁₀₃ is F or S; R; and X₁₀₆ is H or Y X₁₀₄ is A, D, G, H, I, L, T, or V;X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ (SEQ ID NO: 277), X₁₀₅ is H, I, N, R, S,or T; and wherein: X₉₉ is L or V; X₁₀₀ is G, H, N, or S; X₁₀₁ is G; X₁₀₆is H, L, M, R, V, or Y X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A or D; X₁₀₅ is N;and X₁₀₆ is H X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ (SEQ ID NO: 278), wherein:X₉₉ is L or V; X₁₀₀ is S; X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A orD; X₁₀₅ is N; and X₁₀₆ is H X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ (SEQ ID NO:279), wherein: X₉₉ is L; X₁₀₀ is N or S; X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ isS; X₁₀₄ is A or D; X₁₀₅ is N or R; and X₁₀₆ is H

TABLE 10C uPA and/or Matriptase Cleavable Core CM Consensus Sequence 13Core CM Consensus 13 Subgenus of Core CM Consensus 13X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈ X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈ (SEQID NO: 281), (SEQ ID NO: 280), wherein: wherein: X₁₁₁ is C or R; X₁₁₂ isI, S, or Y; X₁₁₃ is G or X₁₁₁ is C, G, H, L, P, R, S, T, or V; R; X₁₁₄is R or S; X₁₁₅ is F, P, or S; X₁₁₆ is D, G, or X₁₁₂ is I, L, M, N, S,T, V, or Y; H; X₁₁₇ is H or N; and X₁₁₈ is H X₁₁₃ is A, D, E, G, K, R,or V; X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈ (SEQ ID NO: 282), X₁₁₄ is A, C,G, H, L, R, S, T, or V; wherein: X₁₁₁ is R; X₁₁₂ is I; X₁₁₃ is G; X₁₁₄is R; X₁₁₅ X₁₁₅ is C, F, P, S, T, V, or Y; is S; X₁₁₆ is D or H; X₁₁₇ isN; and X₁₁₈ is H X₁₁₆ is A, D, E, G, H, N, T, V, or Y; X₁₁₇ is D, E, H,K, N, Q, R, S, T; and X₁₁₈ is H, L, N, R, S, V, or Y

TABLE 10D uPA and/or Matriptase Cleavable Core CM Consensus Sequence 14Core CM Consensus 14 Subgenus of Core CM Consensus 14X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀ X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀ (SEQID NO: 284), (SEQ ID NO: 283), wherein: wherein: X₁₂₃ is L or T; X₁₂₄ isE, R, or S; X₁₂₅ is G; X₁₂₃ is L, R, T, or V; X₁₂₆ is R; X₁₂₇ is P or S;X₁₂₈ is A, G, or Y; X₁₂₉ is E or X₁₂₄ is E, G, I, N, R, or S; N; andX₁₃₀ is P, Q, or S X₁₂₅ is G; X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀ (SEQ IDNO: 285), X₁₂₆ is R; wherein: X₁₂₃ is L, T, or V; X₁₂₄ is R or S; X₁₂₅is G; X₁₂₇ is P or S; X₁₂₆ is R; X₁₂₇ is S; X₁₂₈ is A or G; X₁₂₉ is K, Nor Y; X₁₂₈ is A, G, or Y; and X₁₃₀ is P X₁₂₉ is E, K, N, or Y; andX₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀ (SEQ ID NO: 286), X₁₃₀ is P, Q, or Swherein: X₁₂₃ is L or T; X₁₂₄ is S; X₁₂₅ is G; X₁₂₆ is R; X₁₂₇ is S;X₁₂₈ is A or G; X₁₂₉ is N; and X₁₃₀ is P

In some embodiments, the motif sequence is a substrate for at least uPAand/or matriptase and includes an expanded consensus sequence based onone of the core CM consensus sequence shown in Tables 10A-10D. In someembodiments, the expanded consensus sequence is a consensus sequenceshown in Tables 11A-11D below.

TABLE 11A uPA and/or Matriptase Cleavable Expanded Core CM ConsensusSequence 11 Expanded Core CM Consensus 11 Subgenus of Expanded Core CMConsensus 11 X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ (SEQ ID X₉₅ X₉₆ (SEQ ID NO: 287),wherein: NO: 288), wherein: X₈₅ is A, D, G, K, L, N, R, S, T, X₈₅ is A,D, G, K, N, S, or V; X₈₆ is A, G, K, M, R, S, or or V; T; X₈₇ is D, L,S, or V; X₈₈ is C, G, N, R, S, or T; X₈₉ is X₈₆ is A, G, K, M, P, Q, R,S, or D, G, or S; X₉₀ is R; X₉₁ is F or S; X₉₂ is A, G, I, L, T, T; orV; X₉₃ is H, I, N, or S; X₉₄ is H, M, R, or Y; X₉₅ is E, X₈₇ is D, I, L,R, S, or V; G, K, or R; and X₉₆ is K, R, or S X₈₈ is C, G, H, I, K, N,R, S, T, or X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ X₉₅X₉₆ (SEQ ID Y; NO: 289),wherein: X₈₉ is D, G, or S; X₈₅ is A, D, G, N, R, or T; X₈₆ is G, K, P,R, S, or T; X₈₇ X₉₀ is R; is L or V; X₈₈ is G, H, K, N, S, or T; X₈₉ isG; X₉₀ is R; X₉₁ is F or S; X₉₁ is S; X₉₂ is A or D; X₉₃ is N or R; X₉₄is H or Y; X₉₅ X₉₂ is A, D, G, H, I, L, T, or V; is E, K, N, Q, or R;and X₉₆ is A, K, or R X₉₃ is H, I, N, R, S, or T;X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ X₉₅X₉₆ (SEQ ID X₉₄ is H, L, M, R, V or Y;NO: 290), wherein: X₉₅ is E, G, K, N, Q, R, or V; and X₈₅ is A, D, G, orR; X₈₆ is K, P, R or T; X₈₇ is L or V; X₉₆ is A, G, K, L, Q, R, or S X₈₈is G, H, N or S; X₈₉ is G; X₉₀ is R; X₉₁ is S; X₉₂ is A or D; X₉₃ is N;X₉₄ is H; X₉₅ is K, N, or R; and X₉₆ is A, K, or RX₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ X₉₅X₉₆ (SEQ ID NO: 291), wherein: X₈₅ isA or D; X₈₆ is K, P, or R; X₈₇ is L or V; X₈₈ is S; X₈₉ is G; X₉₀ is R;X₉₁ is S; X₉₂ is A or D; X₉₃ is N; X₉₄ is H; X₉₅ is K or R; and X₉₆ is Kor R X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂X₉₃X₉₄ X₉₅X₉₆ (SEQ ID NO: 292), wherein:X₈₅ is D, G, or N; X₈₆ is K, R, or S; X₈₇ is L or V; X₈₈ is G or S; X₈₉is G; X₉₀ is R; X₉₁ is S; X₉₂ is A; X₉₃ is N; X₉₄ is H; X₉₅ is K; andX₉₆ is K

TABLE 11B uPA and/or Matriptase Cleavable Expanded Core CM ConsensusSequence 12 Expanded Core CM Consensus 12 Subgenus of Expanded Core CMConsensus 12 X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ X₁₀₇X₁₀₈ X₁₀₆X₁₀₇X₁₀₈ (SEQ ID NO:293), (SEQ ID NO: 294), wherein: X₉₇ is D, G, K, or R; X₉₈ wherein: isG, P, or R; X₉₉ is L; X₁₀₀ is N, S, or T; X₁₀₁ is G; X₉₇ is A, D, G, K,L, N, R, S, T, X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A, D, or H; X₁₀₅ is N or R;or V; X₁₀₆ is H, L, V, or Y; X₁₀₇ is E, G, K, N, Q, or R; and X₉₈ is A,G, K, M, P, Q, R, S, or T; X₁₀₈ is A, G, K, L, Q, or R X₉₉ is D, I, L,R, S, or V; X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ X₁₀₇X₁₀₈ X₁₀₀ is C, G,H, I, K, N, R, S, T (SEQ ID NO: 295), wherein: X₉₇ is A, D, G, N, R, oror Y; T; X₉₈ is G, K, P, R, S, or T; X₉₉ is L or V; X₁₀₀ is G, X₁₀₁ isD, G, or S; H, K, N, S, or T; X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ isX₁₀₂ is R; A or D; X₁₀₅ is N or R; X₁₀₆ is H or Y; X₁₀₇ is E, K, N, X₁₀₃is F or S; Q, or R; and X₁₀₈ is A, K, or R X₁₀₄ is A, D, G, H, I, L, T,or V; X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ X₁₀₇X₁₀₈ X₁₀₅ is H, I, N, R,S, or T; (SEQ ID NO: 296), wherein: X₉₇ is A, D, G, or R; X₉₈ X₁₀₆ is H,L, M, R, V, or Y; is K, P, R, or T; X₉₉ is L or V; X₁₀₀ is G, H, N, orS; X₁₀₇ is E, G, K, N, Q, R, or V; and X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ is S;X₁₀₄ is A or D; X₁₀₅ is N; X₁₀₈ is A, G, K, L, Q, R, or S X₁₀₆ is H;X₁₀₇ is K, N, or R; and X₁₀₈ is A, K, or RX₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆ X₁₀₇X₁₀₈ (SEQ ID NO: 297),wherein: X₉₇ is A or D; X₉₈ is K, P, or R; X₉₉ is L or V; X₁₀₀ is S;X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A or D; X₁₀₅ is N; X₁₀₆ is H;X₁₀₇ is K or R; and X₁₀₈ is K or R X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈ (SEQ ID NO: 298), wherein: X₉₇ is G or R; X₉₈ is P; X₉₉ is L;X₁₀₀ is N or S; X₁₀₁ is G; X₁₀₂ is R; X₁₀₃ is S; X₁₀₄ is A or D; X₁₀₅ isN or R; X₁₀₆ is H; X₁₀₇ is K, Q, or R; and X₁₀₈ is A, K, or R

TABLE 11C uPA and/or Matriptase Cleavable Expanded Core CM ConsensusSequence 13 Expanded Core CM Consensus 13 Subgenus of Expanded Core CMConsensus 13 X₁₀₉X₁₁₀X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₀₉X₁₁₀X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀ (SEQ X₁₁₈X₁₁₉X₁₂₀ (SEQID NO: 299), ID NO: 300), wherein: X₁₀₉ is N; X₁₁₀ is H or R; X₁₁₁wherein: is C or R; X₁₁₂ is I, S, or Y; X₁₁₃ is G or R; X₁₁₄ is X₁₀₉ isA, D, G, H, I, K, N, R, S, T, R or S; X₁₁₅ is F, P, or S; X₁₁₆ is D, G,or H; X₁₁₇ is H or Y; or N; X₁₁₈ is H; X₁₁₉ is E, K, R, or V; and X₁₂₀is A, G, X₁₁₀ is D, G, H, L, N, Q, R, or Y; Q, R, or W X₁₁₁ is C, G, H,L, P, R, S, T, or V; X₁₀₉X₁₁₀X₁₁₁X₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈X₁₁₉X₁₂₀X₁₁₂ is I, L, M, N, S, T, V, or Y; (SEQ ID NO: 301), wherein: X₁₀₉ is N;X₁₁₀ is H; X₁₁₁ is X₁₁₃ is A, D, E, G, K, R, or V; R; X₁₁₂ is I; X₁₁₃ isG; X₁₁₄ is R; X₁₁₅ is S; X₁₁₆ is D X₁₁₄ is A, C, G, H, L, R, S, T, or V;or H; X₁₁₇ is N; X₁₁₈ is H; X₁₁₉ is R; and X₁₂₀ is G or R X₁₁₅ is C, F,P, S, T, V, or Y; X₁₁₆ is A, D, E, G, H, N, T, V, or Y; X₁₁₇ is D, E, H,K, N, Q, R, S, T; X₁₁₈ is H, L, N, R, S, V, or Y; X₁₁₉ is E, G, K, L, N,Q, R, S, V, or W; and X₁₂₀ is A, E, G, K, L, N, P, Q, R, or W

TABLE 11D uPA and/or Matriptase Cleavable Expanded Core CM ConsensusSequence 14 Expanded Core CM Consensus 14 Subgenus of Expanded Core CMConsensus 14 X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂ X₁₃₀X₁₃₁X₁₃₂ (SEQ IDNO: 302), (SEQ ID NO: 303), wherein: X₁₂₁ is M, N, P, R, or T; wherein:X₁₂₂ is A, P, or S; X₁₂₃ is L or T; X₁₂₄ is E, R, or S; X₁₂₅ X₁₂₁ is A,D, G, M, N, P, R, or T; is G; X₁₂₆ is R; X₁₂₇ is P or S; X₁₂₈ is A, G,or Y; X₁₂₉ X₁₂₂ is A, H, K, P, R, or S; is E or N; X₁₃₀ is P, Q, or S;X₁₃₁ is E, K, R; and X₁₃₂ is X₁₂₃ is L, R, T, or V; E, G, or R X₁₂₄ isE, G, I, N, R, or S; X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₂₅ is G; (SEQ ID NO: 304), wherein: X₁₂₁ is G, N, or T; X₁₂₂ is X₁₂₆is R; A, P, or S; X₁₂₃ is L, T, or V; X₁₂₄ is R or S; X₁₂₅ is G; X₁₂₇ isP or S; X₁₂₆ is R; X₁₂₇ is S; X₁₂₈ is A or G; X₁₂₉ is K, N or Y; X₁₂₈ isA, G, or Y; X₁₃₀ is P; X₁₃₁ is K or R; and X₁₃₂ is D, G or H X₁₂₉ is E,K, N, or Y; X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂ X₁₃₀ is P,Q, or S; (SEQ ID NO: 305), wherein: X₁₂₁ is T; X₁₂₂ is P or S; X₁₃₁ isE, K, or R; and X₁₂₃ is L or T; X₁₂₄ is S; X₁₂₅ is G; X₁₂₆ is R; X₁₂₇ isS; X₁₃₂ is D, E, G, H, or R X₁₂₈ is A or G; X₁₂₉ is N; X₁₃₀ is P; X₁₃₁is K or R; and X₁₃₂ is G or HX₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆X₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂ (SEQ ID NO: 306),wherein: X₁₂₁ is T; X₁₂₂ is S; X₁₂₃ is L or T; X₁₂₄ is S; X₁₂₅ is G;X₁₂₆ is R; X₁₂₇ is S; X₁₂₈ is A or G; X₁₂₉ is N; X₁₃₀ is P; X₁₃₁ is R;and X₁₃₂ is G

In some embodiments, the CM comprises a core CM consensus 11 sequencecomprising the amino acid sequence LSGRSANH (SEQ ID NO: 307) or LSGRSGNH(SEQ ID NO: 308). In some embodiments, the CM comprises an expanded coreCM consensus 11 sequence comprising the amino acid sequence DRLSGRSANHKK(SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), or NTLSGRSGNHGS (SEQ IDNO: 311).

In some embodiments, the CM comprises the amino acid sequence LSGRSANH(SEQ ID NO: 307). In some embodiments, the CM comprises the amino acidsequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CMcomprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In someembodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQID NO: 310). In some embodiments, the CM comprises the amino acidsequence NTLSGRSGNHGS (SEQ ID NO: 311).

In some embodiments, the CM comprises the amino acid sequence LSGRSANH(SEQ ID NO: 307). In some embodiments, the CM comprises the amino acidsequence LNGRSDNH (SEQ ID NO: 313). In some embodiments, the CMcomprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In someembodiments, the CM comprises a core CM consensus 12 sequence comprisingthe amino acid sequence LSGRSANH (SEQ ID NO: 307), LNGRSDNH (SEQ ID NO:313), and LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM anexpanded core CM consensus 12 sequence comprising an amino acid sequenceselected from the group consisting of DRLSGRSANHKK (SEQ ID NO: 309),DRLSGRSDNHKK (SEQ ID NO: 310), GPLNGRSDNHKA (SEQ ID NO: 320),GPLNGRSDNHKK (SEQ ID NO: 321), GPLNGRSDNHKR (SEQ ID NO: 322),GPLNGRSDNHQA (SEQ ID NO: 323), GPLNGRSDNHQK (SEQ ID NO: 324),GPLNGRSDNHQR (SEQ ID NO: 325), GPLNGRSDNHRA (SEQ ID NO: 326),GPLNGRSDNHRK (SEQ ID NO: 327), GPLNGRSDNHRR (SEQ ID NO: 328),RPLNGRSDNHKA (SEQ ID NO: 329), RPLNGRSDNHKK (SEQ ID NO: 330),RPLNGRSDNHKR (SEQ ID NO: 331), RPLNGRSDNHQA (SEQ ID NO: 332),RPLNGRSDNHQK (SEQ ID NO: 333), RPLNGRSDNHQR (SEQ ID NO: 334),RPLNGRSDNHRA (SEQ ID NO: 335), RPLNGRSDNHRK (SEQ ID NO: 336),RPLNGRSDNHRR (SEQ ID NO: 337), GPLSGRSDNHKA (SEQ ID NO: 338),GPLSGRSDNHKK (SEQ ID NO: 339), GPLSGRSDNHKR (SEQ ID NO: 340),GPLSGRSDNHQA (SEQ ID NO: 341), GPLSGRSDNHQK (SEQ ID NO: 342),GPLSGRSDNHQR (SEQ ID NO: 343), GPLSGRSDNHRA (SEQ ID NO: 344),GPLSGRSDNHRK (SEQ ID NO: 345), GPLSGRSDNHRR (SEQ ID NO: 346),RPLSGRSDNHKA (SEQ ID NO: 347), RPLSGRSDNHKK (SEQ ID NO: 348),RPLSGRSDNHKR (SEQ ID NO: 349), RPLSGRSDNHQA (SEQ ID NO: 350),RPLSGRSDNHQK (SEQ ID NO: 351), RPLSGRSDNHQR (SEQ ID NO: 352),RPLSGRSDNHRA (SEQ ID NO: 353), RPLSGRSDNHRK (SEQ ID NO: 354),RPLSGRSDNHRR (SEQ ID NO: 355), and KGLTGRSDRHQA (SEQ ID NO: 356).

In some embodiments, the CM comprises the amino acid sequenceDRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises theamino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHKA (SEQ ID NO: 320).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHKK (SEQ ID NO: 321). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHKR (SEQ ID NO: 322). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHQA (SEQ ID NO: 323).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHQK (SEQ ID NO: 324). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHQR (SEQ ID NO: 325). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHRA (SEQ ID NO: 326).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHRK (SEQ ID NO: 327). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHRR (SEQ ID NO: 328). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHKA (SEQ ID NO: 329).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHKK (SEQ ID NO: 330). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHKR (SEQ ID NO: 331). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHQA (SEQ ID NO: 332).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHQK (SEQ ID NO: 333). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHQR (SEQ ID NO: 334). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHRA (SEQ ID NO: 335).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHKK (SEQ ID NO: 336). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHKR (SEQ ID NO: 337). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 338).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHKK (SEQ ID NO: 339). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHKR (SEQ ID NO: 340). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHQA (SEQ ID NO: 341).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHQK (SEQ ID NO: 342). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHQR (SEQ ID NO: 343). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 344).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHKK (SEQ ID NO: 345). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHKR (SEQ ID NO: 346). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHKA (SEQ ID NO: 347).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHKK (SEQ ID NO: 348). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHKR (SEQ ID NO: 349). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHQA (SEQ ID NO: 350).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHQK (SEQ ID NO: 351). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHQR (SEQ ID NO: 352). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHRA (SEQ ID NO: 353).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHRK (SEQ ID NO: 354). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHRR (SEQ ID NO: 355). In some embodiments,the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 356).

In some embodiments, the CM comprises a core CM consensus 13 sequencecomprising the amino acid sequence RIGRSDNH (SEQ ID NO: 357) or RLGRSDNN(SEQ ID NO: 358). In some embodiments, the CM comprises an expanded coreCM consensus 13 sequence comprising the amino acid sequence NHRIGRSDNHRR(SEQ ID NO: 359) or TLRLGRSDNNKN (SEQ ID NO: 360).

In some embodiments, the CM comprises the amino acid sequence RIGRSDNH(SEQ ID NO: 357). In some embodiments, the CM comprises the amino acidsequence RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CMcomprises the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359). In someembodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQID NO: 360).

In some embodiments, the CM comprises a core CM consensus 14 sequencecomprising an amino acid sequence selected from the group consisting ofTSGRSANP (SEQ ID NO: 361), TSGRSGNP (SEQ ID NO: 362), LSGRSANP (SEQ IDNO: 363), and LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CMcomprises an expanded core CM consensus 14 sequence comprising an aminoacid sequence selected from the group consisting of TSTSGRSANPRG (SEQ IDNO: 365), TSTSGRSGNPRG (SEQ ID NO: 366), TSLSGRSANPRG (SEQ ID NO: 367),and TSLSGRSGNPRG (SEQ ID NO: 368).

In some embodiments, the CM comprises the amino acid sequence TSGRSANP(SEQ ID NO: 361). In some embodiments, the CM comprises the amino acidsequence TSGRSGNP (SEQ ID NO: 362). In some embodiments, the CMcomprises the amino acid sequence LSGRSANP (SEQ ID NO: 363). In someembodiments, the CM comprises the amino acid sequence LSGRSGNP (SEQ IDNO: 364). In some embodiments. In some embodiments, the CM comprises theamino acid sequence the CM comprises the amino acid sequenceTSTSGRSANPRG (SEQ ID NO: 365). In some embodiments, the CM comprises theamino acid sequence TSTSGRSGNPRG (SEQ ID NO: 366). In some embodiments,the CM comprises the amino acid sequence TSLSGRSANPRG (SEQ ID NO: 367).In some embodiments, the CM comprises the amino acid sequence andTSLSGRSGNPRG (SEQ ID NO: 368).

In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of LSGRSENH (SEQ ID NO: 369), SIARSDNL (SEQ IDNO: 370), LSGRSVTQ (SEQ ID NO: 371), LSGRSGNH (SEQ ID NO: 308), LTGRSDRH(SEQ ID NO: 314), LYGRSENN (SEQ ID NO: 374), RLGRSDNN (SEQ ID NO: 375),TSGRSANP (SEQ ID NO: 376), NTLSGRSENHSG (SEQ ID NO: 377), PPSIARSDNLAN(SEQ ID NO: 378), TGLSGRSVTQTS (SEQ ID NO: 379), NTLSGRSGNHGS (SEQ IDNO: 311), KGLTGRSDRHQA (SEQ ID NO: 381), KNLYGRSENNGN (SEQ ID NO: 382),TLRLGRSDNNKN (SEQ ID NO: 383), and TSTSGRSANPRG (SEQ ID NO: 384).

In some embodiments, the CM comprises the amino acid sequence LSGRSENH(SEQ ID NO: 369). In some embodiments, the CM comprises the amino acidsequence SIARSDNL (SEQ ID NO: 370). In some embodiments, the CMcomprises the amino acid sequence LSGRSVTQ (SEQ ID NO: 371). In someembodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ IDNO: 308). In some embodiments, the CM comprises the amino acid sequenceLTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises theamino acid sequence LYGRSENN (SEQ ID NO: 374). In some embodiments, theCM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 375). In someembodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ IDNO: 376). In some embodiments, the CM comprises the amino acid sequenceNTLSGRSENHSG (SEQ ID NO: 377). In some embodiments, the CM comprises theamino acid sequence PPSIARSDNLAN (SEQ ID NO: 378). In some embodiments,the CM comprises the amino acid sequence TGLSGRSVTQTS (SEQ ID NO: 379).In some embodiments, the CM comprises the amino acid sequenceNTLSGRSGNHGS (SEQ ID NO: 311). In some embodiments, the CM comprises theamino acid sequence KGLTGRSDRHQA (SEQ ID NO: 381). In some embodiments,the CM comprises the amino acid sequence KNLYGRSENNGN (SEQ ID NO: 382).In some embodiments, the CM comprises the amino acid sequenceTLRLGRSDNNKN (SEQ ID NO: 383). In some embodiments, the CM comprises theamino acid sequence TSTSGRSANPRG (SEQ ID NO: 384).

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, at least one protease is matriptase or uPA and atleast one protease is selected from the group consisting of those shownin Table 7.

TABLE 7 Exemplary Proteases and/or Enzymes ADAMS, ADAMTS, e.g. ADAM8ADAM9 ADAM10 ADAM12 ADAM15 ADAM17/TACE ADAMDEC1 ADAMTS1 ADAMTS4 ADAMTS5Aspartate proteases, e.g., BACE Renin Aspartic cathepsins, e.g.,Cathepsin D Cathepsin E Caspases, e.g., Caspase 1 Caspase 2 Caspase 3Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10Caspase 14 Cysteine cathepsins, e.g., Cathepsin B Cathepsin C CathepsinK Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/P Cysteineproteinases, e.g., Cruzipain Legumain Otubain-2 KLKs, e.g., KLK4 KLK5KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g., MeprinNeprilysin PSMA BMP-1 MMPs, e.g., MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP23 MMP24 MMP26MMP27 Serine proteases, e.g., activated protein C Cathepsin A CathepsinG Chymase coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa,FXIIa) Elastase Granzyme B Guanidinobenzoatase HtrA1 Human NeutrophilElastase Lactoferrin Marapsin NS3/4A PACE4 Plasmin PSA tPA ThrombinTryptase uPA Type II Transmembrane Serine Proteases (TTSPs), e.g., DESC1DPP-4 FAP Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2 TMPRSS3 TMPRSS4

In some embodiments, the antibody is attached to at least a first CM anda second CM. In some embodiments, the first CM and the second CM areeach polypeptides of no more than 15 amino acids long. In someembodiments, the first CM and the second CM in the antibody in theuncleaved state have the structural arrangement from N-terminus toC-terminus as follows: Agent-CM1-CM2-(Antibody or Antigen-BindingFragment), (Antibody or Antigen-Binding Fragment)-CM2-CM1-Agent,Agent-CM2-CM1-(Antibody or Antigen-Binding Fragment), or (Antibody orAntigen-Binding Fragment)-CM1-CM2-Agent. In some embodiments, theantibody includes a linking peptide between the agent and CM1. In someembodiments, the antibody includes a linking peptide between CM1 andCM2. In some embodiments, the antibody includes a linking peptidebetween CM2 and antibody or antigen-binding fragment. In someembodiments, the antibody includes a linking peptide between the agentand CM1 and a linking peptide between CM2 and antibody orantigen-binding fragment. In some embodiments, the antibody includes alinking peptide between agent and CM1 and a linking peptide between CM1and CM2. In some embodiments, the antibody includes a linking peptidebetween CM1 and CM2 and a linking peptide between CM2 and antibody orantigen-binding fragment. In some embodiments, the antibody includes alinking peptide between agent and CM1, a linking peptide between CM1 andCM2, and a linking peptide between CM2 and antibody or antigen-bindingfragment.

In some embodiments, the antibody includes at least a first CM thatincludes a substrate for at least one protease selected from matriptaseand uPA and a second CM that includes a substrate sequence. Exemplarysubstrates for the second CM (CM2) include but are not limited tosubstrates cleavable by one or more of the following enzymes orproteases listed in Table 7.

In some embodiments, the CM2 is selected for use with a specificprotease. In some embodiments, the CM2 is a substrate for at least oneprotease selected from the group consisting of a matrix metalloprotease(MMP), a neutrophil elastase, uPA (also referred to as u-plasminogenactivator), legumain, matriptase, thrombin, a cysteine protease such asa cathepsin, ADAM17, BMP-1, HtrA1, and a TMPRSS such as TMPRSS3 orTMPRSS4.

In some embodiments, the CM2 is a substrate for a neutrophil elastase.In some embodiments, the CM2 is a substrate for uPA. In someembodiments, the CM2 is a substrate for legumain. In some embodiments,the CM2 is a substrate for matriptase. In some embodiments, the CM2 is asubstrate for thrombin. In some embodiments, the CM2 is a substrate fora cysteine protease. In some embodiments, the CM2 is a substrate for acathepsin. In some embodiments, the CM2 is a substrate for ADAM17. Insome embodiments, the CM2 is a substrate for BMP-1. In some embodiments,the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is asubstrate for a TMPRSS. In some embodiments, the CM2 is a substrate forTMPRSS3. In some embodiments, the CM2 is a substrate for TMPRSS4.

For example, suitable CM2 are cleaved by at least one protease andinclude the sequence TGRGPSWV (SEQ ID NO: 402); SARGPSRW (SEQ ID NO:403); TARGPSFK (SEQ ID NO: 404); TARGPSW (SEQ ID NO: 405); LSGRSDNH (SEQID NO: 406); GGWHTGRN (SEQ ID NO: 407); HTGRSGAL (SEQ ID NO: 408);PLTGRSGG (SEQ ID NO: 409); AARGPAIH (SEQ ID NO: 411); RGPAFNPM (SEQ IDNO: 412); SSRGPAYL (SEQ ID NO: 413); RGPATPIM (SEQ ID NO: 414); RGPA(SEQ ID NO: 415); GGQPSGMWGW (SEQ ID NO: 416); FPRPLGITGL (SEQ ID NO:417); VHMPLGFLGP (SEQ ID NO: 418); SPLTGRSG (SEQ ID NO: 419); SAGFSLPA(SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 420); SGGPLGVR (SEQ ID NO: 421);PLGL (SEQ ID NO: 422); GPRSFGL (SEQ ID NO: 423) and/or GPRSFG (SEQ IDNO: 424).

In some embodiments, the CM2 comprises the amino acid sequence TGRGPSWV(SEQ ID NO: 402). In some embodiments, the CM2 comprises the amino acidsequence SARGPSRW (SEQ ID NO: 403). In some embodiments, the CM2comprises the amino acid sequence TARGPSFK (SEQ ID NO: 404). In someembodiments, the CM2 comprises the amino acid sequence TARGPSW (SEQ IDNO: 405). In some embodiments, the CM2 comprises the amino acid sequenceLSGRSDNH (SEQ ID NO: 406). In some embodiments, the CM2 comprises theamino acid sequence GGWHTGRN (SEQ ID NO: 407). In some embodiments, theCM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 408). In someembodiments, the CM2 comprises the amino acid sequence PLTGRSGG (SEQ IDNO: 409). In some embodiments, the CM2 comprises the amino acid sequenceAARGPAIH (SEQ ID NO: 411). In some embodiments, the CM2 comprises theamino acid sequence RGPAFNPM (SEQ ID NO: 412). In some embodiments, theCM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 413). In someembodiments, the CM2 comprises the amino acid sequence RGPATPIM (SEQ IDNO: 414). In some embodiments, the CM2 comprises the amino acid sequenceRGPA (SEQ ID NO: 415). In some embodiments, the CM2 comprises the aminoacid sequence GGQPSGMWGW (SEQ ID NO: 416). In some embodiments, the CM2comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 417). In someembodiments, the CM2 comprises the amino acid sequence VHMPLGFLGP (SEQID NO: 418). In some embodiments, the CM2 comprises the amino acidsequence SPLTGRSG (SEQ ID NO: 419). In some embodiments, the CM2comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 420). In someembodiments, the CM2 comprises the amino acid sequence SGGPLGVR (SEQ IDNO: 421). In some embodiments, the CM2 comprises the amino acid sequencePLGL (SEQ ID NO: 422). In some embodiments, the CM2 comprises the aminoacid sequence GPRSFGL (SEQ ID NO: 423). In some embodiments, the CM2comprises the amino acid sequence GPRSFG (SEQ ID NO: 424).

In some embodiments, the CM2 is a substrate for at least one MMP. Insome embodiments, the CM2 is a substrate for at least one MMP listed inthe Table 7. In some embodiments, the CM2 is a substrate for MMP9. Insome embodiments, the CM2 is a substrate for MMP14. In some embodiments,the CM2 is a substrate for two or more MMPs. In some embodiments, theCM2 is a substrate for at least MMP9 or MMP14. In some embodiments, theCM2 is a substrate for two or more MMPs. In some embodiments, the CM2 isa substrate for at least MMP9 and MMP14.

In some embodiments, CM2 is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 425); QNQALRMA (SEQ ID NO: 426); AQNLLGMV(SEQ ID NO: 427); STFPFGMF (SEQ ID NO: 428); PVGYTSSL (SEQ ID NO: 429);DWLYWPGI (SEQ ID NO: 430); MIAPVAYR (SEQ ID NO: 431); RPSPMWAY (SEQ IDNO: 432); WATPRPMR (SEQ ID NO: 433); FRLLDWQW (SEQ ID NO: 434); LKAAPRWA(SEQ ID NO: 435); GPSHLVLT (SEQ ID NO: 436); LPGGLSPW (SEQ ID NO: 437);MGLFSEAG (SEQ ID NO: 438); SPLPLRVP (SEQ ID NO: 439); RMHLRSLG (SEQ IDNO: 440); LAAPLGLL (SEQ ID NO: 441); AVGLLAPP (SEQ ID NO: 442); LLAPSHRA(SEQ ID NO: 443), PAGLWLDP (SEQ ID NO: 444); and/or ISSGLSS (SEQ ID NO:445).

In some embodiments, the first cleaving agent and the second cleavingagent are the same protease, and the first CM and the second CM aredifferent substrates for the enzyme. In some embodiments, the firstcleaving agent and the second cleaving agent are different proteases. Insome embodiments, the first cleaving agent and the second cleaving agentare co-localized in the target tissue. In some embodiments, the first CMand the second CM are cleaved by at least one cleaving agent in thetarget tissue.

In some embodiments, the CM comprises the non-prime side of the proteasecleavage site; that is, the CM comprises at least the P1 and P2 aminoacids, and in some embodiments comprises the P1, P2 and P3 amino acidsand in some embodiments comprises the P1, P2, P3, and P4 amino acids. Insome embodiments, the CM comprises the non-prime side and the prime sideof the protease cleavage site. In some embodiments, the CM comprises thenon-prime side but lacks at least part of the prime side of the proteasecleavage site. In some embodiments, the CM comprises the non-prime sidebut lacks the prime side of the protease cleavage site. Such a CM can belinked directly or through a linker to an antibody or other molecule asdisclosed herein, such as, but not limited to, a detection moiety.

In some embodiments, the agent conjugated to the antibody orantigen-binding fragment is a therapeutic agent. In some embodiments,the agent is an antineoplastic agent. In some embodiments, the agent isa toxin or fragment thereof. As used herein, a fragment of a toxin is afragment that retains toxic activity. In some embodiments, the agent isconjugated to the AB via a cleavable linker. In some embodiments, theagent is conjugated to the AB via a linker that includes at least onecleavable substrate sequence described herein. In some embodiments, theagent is conjugated to the AB via a noncleavable linker. In someembodiments, the agent is a microtubule inhibitor. In some embodiments,the agent is a nucleic acid damaging agent, such as a DNA alkylator orDNA intercalator, or other DNA damaging agent. In some embodiments, theagent is an agent selected from the group listed in Table 3. In someembodiments, the agent is a dolastatin. In some embodiments, the agentis an auristatin or derivative thereof. In some embodiments, the agentis auristatin E or a derivative thereof. In some embodiments, the agentis monomethyl auristatin E (MMAE). In some embodiments, the agent ismonomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof. In some embodiments, the agent is apyrrolobenzodiazepine.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the antibody also includes a detectable moiety. Insome embodiments, the detectable moiety is a diagnostic agent.

In some embodiments, the conjugated antibody and/or conjugatedactivatable antibody includes a detectable label. In some embodiments,the detectable label includes an imaging agent, a contrasting agent, anenzyme, a fluorescent label, a chromophore, a dye, one or more metalions, or a ligand-based label. In some embodiments, the imaging agentcomprises a radioisotope. In some embodiments, the radioisotope isindium or technetium. In some embodiments, the contrasting agentcomprises iodine, gadolinium or iron oxide. In some embodiments, theenzyme comprises horseradish peroxidase, alkaline phosphatase, orβ-galactosidase. In some embodiments, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments, the luminescent label comprises anN-methylacrydium derivative. In some embodiments, the label comprises anAlexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In someembodiments, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments, the antibody naturally contains one or moredisulfide bonds. In some embodiments, the antibody or antigen-bindingfragment can be engineered to include one or more disulfide bonds.

In some embodiments, the antibody and/or conjugated antibody ismonospecific. In some embodiments, the antibody and/or conjugatedantibody is multispecific, e.g., by way of non-limiting example,bispecific or trifunctional. In some embodiments, the antibody and/orconjugated antibody is formulated as part of a pro-Bispecific T CellEngager (pro-BITE) molecule. In some embodiments, the antibody and/orconjugated antibody is formulated as part of a pro-Chimeric AntigenReceptor (pro-CAR) modified T cell or other engineered receptor.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody is monospecific. In some embodiments, theactivatable antibody and/or conjugated activatable antibody ismultispecific, referred to herein as multispecific activatableantibodies and/or conjugated multispecific activatable antibodies. Asused herein, terms such as “activatable antibody” and all grammaticalvariations thereof, unless otherwise noted, are intended to encompass,but are not limited to embodiments where the activatable antibody is amultispecific activatable antibody of the disclosure. As used herein,terms such as “conjugated activatable antibody” and all grammaticalvariations thereof, unless otherwise noted, are intended to encompass,but are not limited to embodiments where the conjugated activatableantibody is a conjugated multispecific activatable antibody of thedisclosure. In some embodiments, the multispecific activatable antibodyand/or conjugated multispecific activatable antibody is bispecific ortrifunctional.

In some embodiments, the conjugated antibodies described herein are usedin conjunction with one or more additional agents or a combination ofadditional agents. Suitable additional agents include currentpharmaceutical and/or surgical therapies for an intended application,such as, for example, cancer. For example, the conjugated antibodies canbe used in conjunction with an additional chemotherapeutic oranti-neoplastic agent.

The matriptase and/or uPA substrates of the disclosure are also usefulin activatable antibodies. The activatable antibodies described hereinin an activated state bind a given target and include (i) an antibody oran antigen binding fragment thereof (AB) that specifically binds to thetarget; (ii) a masking moiety (MM) that inhibits the binding of the ABto the target in an uncleaved state; and (c) a cleavable moiety (CM)coupled to the AB, wherein the CM is a polypeptide that functions as asubstrate for at least one protease selected from matriptase and/or uPA.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the MM and the CM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the CM and the AB.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP1-MM.

In some embodiments, the two linking peptides need not be identical toeach other.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 385) and (GGGS)_(n) (SEQ ID NO: 386), where n isan integer of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 387),GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO: 389), GSGGG (SEQ ID NO: 390),GGGSG (SEQ ID NO: 391), and GSSSG (SEQ ID NO: 392).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 393), GSSGGSGGSGG (SEQ ID NO: 394), GSSGGSGGSGGS (SEQ ID NO:395), GSSGGSGGSGGSGGGS (SEQ ID NO: 396), GSSGGSGGSG (SEQ ID NO: 397), orGSSGGSGGSGS (SEQ ID NO: 398).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS,GGGS (SEQ ID NO: 399), GSSGT (SEQ ID NO: 400) or GSSG (SEQ ID NO: 401).

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to the target.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds a target. Insome embodiments, the antibody or immunologically active fragmentthereof that binds the target is a monoclonal antibody, domain antibody,single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, asingle domain heavy chain antibody, or a single domain light chainantibody. In some embodiments, such an antibody or immunologicallyactive fragment thereof that binds the target is a mouse, other rodent,chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is no more than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM does not interfere or compete with the ABfor binding to the target in a cleaved state.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length. For example, the MM is a polypeptide of up to about 40amino acids in length.

In some embodiments, the MM polypeptide sequence is different from thatof any natural binding partner of the AB. In some embodiments, the MMpolypeptide sequence is no more than 50% identical to any naturalbinding partner of the AB. In some embodiments, the MM polypeptidesequence is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical toany natural binding partner of the AB.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leasttwo times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leastthree times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at leastfive times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least10 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind its target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least20 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least40 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least100 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least1000 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the coupling of the MM to the AB reduces theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least10,000 times greater than the K_(d) of the AB when not coupled to the MMtowards the target.

In some embodiments, the protease, i.e., matriptase and/or uPA, isco-localized with the target in a tissue, and the protease cleaves theCM in the activatable antibody when the activatable antibody is exposedto the protease.

In some embodiments, in the presence of the target, the MM reduces theability of the AB to bind the target by at least 90% when the CM isuncleaved, as compared to when the CM is cleaved when assayed in vitrousing a target displacement assay such as, for example, the assaydescribed in PCT Publication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least twofold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least five-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least ten-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 20-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state (i.e., when the activatable antibody is in the cleavedstate), the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 40-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 50-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 100-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 200-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, whereas in thecleaved state, the AB binds the target.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM is a substrate for at least matriptase. Insome embodiments, the CM is a substrate for at least uPA. In someembodiments, the CM is a substrate for at least matriptase and uPA.

In some embodiments, the CM is a substrate for matriptase and/or uPA,and is resistant to cleavage by at least one other protease. In someembodiments, the CM is a substrate for matriptase and/or uPA, and isresistant to cleavage by at least plasmin. In some embodiments, the CMis a substrate for matriptase and/or uPA, and is resistant to cleavageby at least tissue plasminogen activator (tPA).

In some embodiments, the CM is a substrate for matriptase and/or uPA andincludes a motif sequence that is recognized by matriptase and/or uPA,provided that for any given motif sequence of the disclosure:

-   -   (i) the CM does not comprise any of the following amino acid        sequences TGRGPSWV (SEQ ID NO: 402), SARGPSRW (SEQ ID NO: 403),        or TARGPSFK (SEQ ID NO: 404); and the CM does not comprise a        consensus amino acid sequence based on these amino acid        sequences, such as for example, TARGPSW (SEQ ID NO: 405);    -   (ii) the CM does not comprise any of the following amino acid        sequences LSGRSDNH (SEQ ID NO: 406), GGWHTGRN (SEQ ID NO: 407),        HTGRSGAL (SEQ ID NO: 408), or PLTGRSGG (SEQ ID NO: 409); and the        CM does not comprise a consensus amino acid sequence based on        these amino acid sequences, such as for example, LTGRSGA (SEQ ID        NO: 410); and/or    -   (iii) the CM does not comprise any of the following amino acid        sequences AARGPAIH (SEQ ID NO: 411), RGPAFNPM (SEQ ID NO: 412),        SSRGPAYL (SEQ ID NO: 413), or RGPATPIM (SEQ ID NO: 414); and the        CM does not comprise a consensus amino acid sequence based on        these amino acid sequences, such as for example, RGPA (SEQ ID        NO: 415).

In some embodiments, the motif sequence includes a core CM consensussequence shown in Tables 8A-8J. In some embodiments, the motif sequenceincludes a subgenus, i.e., a subset, of the core CM consensus sequenceshown in Tables 8A-8J.

In some embodiments, the motif sequence includes an expanded consensussequence based on one of the core CM consensus sequence shown in Tables8A-8J. In some embodiments, the expanded consensus sequence is aconsensus sequence shown in Tables 9A-9J-3.

In some embodiments, the CM comprises a core CM consensus 1 sequencecomprising the amino acid sequence AAPRS (SEQ ID NO: 163). In someembodiments, the CM comprises an expanded core CM consensus 1 sequencecomprising the amino acid sequence AAPRSF (SEQ ID NO: 164).

In some embodiments, the CM comprises a core CM consensus 2 sequencecomprising the amino acid sequence SRRVP (SEQ ID NO: 165). In someembodiments, the CM comprises an expanded core CM consensus 2 sequencecomprising an amino acid sequence selected from the group consisting ofQSRRVP (SEQ ID NO: 166), QTRRVP (SEQ ID NO: 167), SRRVPL (SEQ ID NO:168), SRRVPV (SEQ ID NO: 169), QSRRVPL (SEQ ID NO: 170), QSRRVPV (SEQ IDNO: 171), QTRRVPL (SEQ ID NO: 172), and QTRRVPV (SEQ ID NO: 173).

In some embodiments, the CM comprises the amino acid sequence QSRRVP(SEQ ID NO: 166). In some embodiments, the CM comprises the amino acidsequence QTRRVP (SEQ ID NO: 167). In some embodiments, the CM comprisesthe amino acid sequence SRRVPL (SEQ ID NO: 168). In some embodiments,the CM comprises the amino acid sequence SRRVPV (SEQ ID NO: 169). Insome embodiments, the CM comprises the amino acid sequence QSRRVPL (SEQID NO: 170). In some embodiments, the CM comprises the amino acidsequence QSRRVPV (SEQ ID NO: 171). In some embodiments, the CM comprisesthe amino acid sequence QTRRVPL (SEQ ID NO: 172). In some embodiments,the CM comprises the amino acid sequence QTRRVPV (SEQ ID NO: 173).

In some embodiments, the CM comprises a core CM consensus 3 sequencecomprising the amino acid sequence PPLGR (SEQ ID NO: 174). In someembodiments, the CM comprises an expanded core CM consensus 3 sequencecomprising an amino acid sequence selected from the group consisting ofGPPLGR (SEQ ID NO: 175), SPPLGR (SEQ ID NO: 176), CGPPLGR (SEQ ID NO:177), CSPPLGR (SEQ ID NO: 178), GGPPLGR (SEQ ID NO: 179), GSPPLGR (SEQID NO: 180), SGPPLGR (SEQ ID NO: 181), SSPPLGR (SEQ ID NO: 182),GCGPPLGR (SEQ ID NO: 183), GCSPPLGR (SEQ ID NO: 184), GGGPPLGR (SEQ IDNO: 185), GGSPPLGR (SEQ ID NO: 186), GSGPPLGR (SEQ ID NO: 187), GSSPPLGR(SEQ ID NO: 188), SCGPPLGR (SEQ ID NO: 189), SCSPPLGR (SEQ ID NO: 190),SGGPPLGR (SEQ ID NO: 191), SGSPPLGR (SEQ ID NO: 192), SSGPPLGR (SEQ IDNO: 193), and SSSPPLGR (SEQ ID NO: 194).

In some embodiments, the CM comprises the amino acid sequence GPPLGR(SEQ ID NO: 175). In some embodiments, the CM comprises the amino acidsequence SPPLGR (SEQ ID NO: 176). In some embodiments, the CM comprisesthe amino acid sequence CGPPLGR (SEQ ID NO: 177). In some embodiments,the CM comprises the amino acid sequence CSPPLGR (SEQ ID NO: 178). Insome embodiments, the CM comprises the amino acid sequence GGPPLGR (SEQID NO: 179). In some embodiments, the CM comprises the amino acidsequence GSPPLGR (SEQ ID NO: 180). In some embodiments, the CM comprisesthe amino acid sequence SGPPLGR (SEQ ID NO: 181). In some embodiments,the CM comprises the amino acid sequence SSPPLGR (SEQ ID NO: 182). Insome embodiments, the CM comprises the amino acid sequence GCGPPLGR (SEQID NO: 183). In some embodiments, the CM comprises the amino acidsequence GCSPPLGR (SEQ ID NO: 184). In some embodiments, the CMcomprises the amino acid sequence GGGPPLGR (SEQ ID NO: 185). In someembodiments, the CM comprises the amino acid sequence GGSPPLGR (SEQ IDNO: 186). In some embodiments, the CM comprises the amino acid sequenceGSGPPLGR (SEQ ID NO: 187). In some embodiments, the CM comprises theamino acid sequence GSSPPLGR (SEQ ID NO: 188). In some embodiments, theCM comprises the amino acid sequence SCGPPLGR (SEQ ID NO: 189). In someembodiments, the CM comprises the amino acid sequence SCSPPLGR (SEQ IDNO: 190). In some embodiments, the CM comprises the amino acid sequenceSGGPPLGR (SEQ ID NO: 191). In some embodiments, the CM comprises theamino acid sequence SGSPPLGR (SEQ ID NO: 192). In some embodiments, theCM comprises the amino acid sequence SSGPPLGR (SEQ ID NO: 193). In someembodiments, the CM comprises the amino acid sequence SSSPPLGR (SEQ IDNO: 194).

In some embodiments, the CM comprises a core CM consensus 4 sequencecomprising the amino acid sequence LRSGW (SEQ ID NO: 195). In someembodiments, the CM comprises an expanded core CM consensus 4 sequencecomprising an amino acid sequence selected from the group consisting ofMLRSGW, (SEQ ID NO: 196), MLRSGWR, (SEQ ID NO: 197), MLRSGWRG, (SEQ IDNO: 198), MLRSGWRL, (SEQ ID NO: 199), and MLRSGWRS (SEQ ID NO: 200).

In some embodiments, the CM comprises the amino acid sequence MLRSGW,(SEQ ID NO: 196). In some embodiments, the CM comprises the amino acidsequence MLRSGWR (SEQ ID NO: 197). In some embodiments, the CM comprisesthe amino acid sequence MLRSGWRG (SEQ ID NO: 198). In some embodiments,the CM comprises the amino acid sequence MLRSGWRL (SEQ ID NO: 199). Insome embodiments, the CM comprises the amino acid sequence MLRSGWRS (SEQID NO: 200).

In some embodiments, the CM comprises a core CM consensus 5 sequencecomprising the amino acid sequence VSRSA (SEQ ID NO: 201). In someembodiments, the CM comprises an expanded core CM consensus 5 sequencecomprising an amino acid sequence selected from the group consisting ofIVSRSA (SEQ ID NO: 202), YIVSRSA (SEQ ID NO: 203), and QYIVSRSA (SEQ IDNO: 204).

In some embodiments, the CM comprises the amino acid sequence IVSRSA(SEQ ID NO: 202). In some embodiments, the CM comprises the amino acidsequence YIVSRSA (SEQ ID NO: 203). In some embodiments, the CM comprisesthe amino acid sequence QYIVSRSA (SEQ ID NO: 204).

In some embodiments, the CM comprises a core CM consensus 6 sequencecomprising the amino acid sequence ALRAP (SEQ ID NO: 205). In someembodiments, the CM comprises an expanded core CM consensus 6 sequencecomprising the amino acid sequence RALRAP (SEQ ID NO: 206).

In some embodiments, the CM comprises a core CM consensus 7 sequencecomprising the amino acid sequence PAGRR (SEQ ID NO: 207). In someembodiments, the CM comprises an expanded core CM consensus 7 sequencecomprising an amino acid sequence selected from the group consisting ofPAGRRS (SEQ ID NO: 208), PAGRRSL (SEQ ID NO: 209), VPAGRRS (SEQ ID NO:210), and VPAGRRSL (SEQ ID NO: 211).

In some embodiments, the CM comprises the amino acid sequence PAGRRS(SEQ ID NO: 208). In some embodiments, the CM comprises the amino acidsequence PAGRRSL (SEQ ID NO: 209). In some embodiments, the CM comprisesthe amino acid sequence VPAGRRS (SEQ ID NO: 210). In some embodiments,the CM comprises the amino acid sequence VPAGRRSL (SEQ ID NO: 211).

In some embodiments, the CM comprises a core CM consensus 8 sequencecomprising the amino acid sequence GRSML (SEQ ID NO: 212). In someembodiments, the CM comprises an expanded core CM consensus 8 sequencecomprising an amino acid sequence selected from the group consisting ofGRSMLL (SEQ ID NO: 213), GRSMLM (SEQ ID NO: 214), GRSMLLG (SEQ ID NO:215), GRSMLLP (SEQ ID NO: 216), GRSMLLS (SEQ ID NO: 217), GRSMLMG (SEQID NO: 218), GRSMLMP (SEQ ID NO: 219), GRSMLMS (SEQ ID NO: 220),GRSMLLGG (SEQ ID NO: 221), GRSMLLPG (SEQ ID NO: 222), GRSMLLSG (SEQ IDNO: 223), GRSMLMGG (SEQ ID NO: 224), GRSMLMPG (SEQ ID NO: 225), GRSMLMSG(SEQ ID NO: 226), GRSMLLGP (SEQ ID NO: 227), GRSMLLPP (SEQ ID NO: 228),GRSMLLSP (SEQ ID NO: 229), GRSMLMGP (SEQ ID NO: 230), GRSMLMPP (SEQ IDNO: 231), GRSMLMSP (SEQ ID NO: 232), GRSMLLGS (SEQ ID NO: 233), GRSMLLPS(SEQ ID NO: 234), GRSMLLSS (SEQ ID NO: 235), GRSMLMGS (SEQ ID NO: 236),GRSMLMPS (SEQ ID NO: 237), and GRSMLMSS (SEQ ID NO: 238).

In some embodiments, the CM comprises the amino acid sequence GRSMLL(SEQ ID NO: 213). In some embodiments, the CM comprises the amino acidsequence GRSMLM (SEQ ID NO: 214). In some embodiments, the CM comprisesthe amino acid sequence GRSMLLG (SEQ ID NO: 215). In some embodiments,the CM comprises the amino acid sequence GRSMLLP (SEQ ID NO: 216). Insome embodiments, the CM comprises the amino acid sequence GRSMLLS (SEQID NO: 217). In some embodiments, the CM comprises the amino acidsequence GRSMLMG (SEQ ID NO: 218). In some embodiments, the CM comprisesthe amino acid sequence GRSMLMP (SEQ ID NO: 219). In some embodiments,the CM comprises the amino acid sequence GRSMLMS (SEQ ID NO: 220). Insome embodiments, the CM comprises the amino acid sequence GRSMLLGG (SEQID NO: 221). In some embodiments, the CM comprises the amino acidsequence GRSMLLPG (SEQ ID NO: 222). In some embodiments, the CMcomprises the amino acid sequence GRSMLLSG (SEQ ID NO: 223). In someembodiments, the CM comprises the amino acid sequence GRSMLMGG (SEQ IDNO: 224). In some embodiments, the CM comprises the amino acid sequenceGRSMLMPG (SEQ ID NO: 225). In some embodiments, the CM comprises theamino acid sequence GRSMLMSG (SEQ ID NO: 226). In some embodiments, theCM comprises the amino acid sequence GRSMLLGP (SEQ ID NO: 227). In someembodiments, the CM comprises the amino acid sequence GRSMLLPP (SEQ IDNO: 228). In some embodiments, the CM comprises the amino acid sequenceGRSMLLSP (SEQ ID NO: 229). In some embodiments, the CM comprises theamino acid sequence GRSMLMGP (SEQ ID NO: 230). In some embodiments, theCM comprises the amino acid sequence GRSMLMPP (SEQ ID NO: 231). In someembodiments, the CM comprises the amino acid sequence GRSMLMSP (SEQ IDNO: 232). In some embodiments, the CM comprises the amino acid sequenceGRSMLLGS (SEQ ID NO: 233). In some embodiments, the CM comprises theamino acid sequence GRSMLLPS (SEQ ID NO: 234). In some embodiments, theCM comprises the amino acid sequence GRSMLLSS (SEQ ID NO: 235). In someembodiments, the CM comprises the amino acid sequence GRSMLMGS (SEQ IDNO: 236). In some embodiments, the CM comprises the amino acid sequenceGRSMLMPS (SEQ ID NO: 237). In some embodiments, the CM comprises theamino acid sequence GRSMLMSS (SEQ ID NO: 238).

In some embodiments, the CM comprises a core CM consensus 9 sequencecomprising the amino acid sequence LARAG (SEQ ID NO: 239). In someembodiments, the CM comprises an expanded core CM consensus 9 sequencecomprising an amino acid sequence selected from the group consisting ofLARAGI (SEQ ID NO: 240), LARAGL (SEQ ID NO: 241), PLARAGI (SEQ ID NO:242), PLARAGL (SEQ ID NO: 243), RPLARAGI (SEQ ID NO: 244), and RPLARAGL(SEQ ID NO: 245).

In some embodiments, the CM comprises the amino acid sequence LARAGI(SEQ ID NO: 240). In some embodiments, the CM comprises the amino acidsequence LARAGL (SEQ ID NO: 241). In some embodiments, the CM comprisesthe amino acid sequence PLARAGI (SEQ ID NO: 242). In some embodiments,the CM comprises the amino acid sequence PLARAGL (SEQ ID NO: 243). Insome embodiments, the CM comprises the amino acid sequence RPLARAGI (SEQID NO: 244). In some embodiments, the CM comprises the amino acidsequence RPLARAGL (SEQ ID NO: 245).

In some embodiments, the CM comprises a core CM consensus 10 sequencecomprising the amino acid sequence ESRRW (SEQ ID NO: 246). In someembodiments, the CM comprises an expanded core CM consensus 10 sequencecomprising an amino acid sequence selected from the group consisting ofESRRWM (SEQ ID NO: 247), ESRRWMP (SEQ ID NO: 248), and PESRRWMP (SEQ IDNO: 249).

In some embodiments, the CM comprises the amino acid sequence ESRRWM(SEQ ID NO: 247). In some embodiments, the CM comprises the amino acidsequence ESRRWMP (SEQ ID NO: 248). In some embodiments, the CM comprisesthe amino acid sequence PESRRWMP (SEQ ID NO: 249).

In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of ILPRSPAF (SEQ ID NO: 250), VAGRSMRP (SEQ IDNO: 251), VVPEGRRS (SEQ ID NO: 252), QGRAITFI (SEQ ID NO: 253), VLSKQMSF(SEQ ID NO: 254), LKGRSYYY (SEQ ID NO: 255), KRMPVQFL (SEQ ID NO: 256),PQHRIVSF (SEQ ID NO: 257), YKKFVGSL (SEQ ID NO: 258), HMMQYARH (SEQ IDNO: 259), IPFSWSRF (SEQ ID NO: 260), LSQARWRK (SEQ ID NO: 261), DISHWRRS(SEQ ID NO: 262), RKTVQHWW (SEQ ID NO: 263), RFYRNQFF (SEQ ID NO: 264),RSLVFAPI (SEQ ID NO: 265), RSPSRLKC (SEQ ID NO: 266), and RKMPNITV (SEQID NO: 267).

In some embodiments, the CM comprises the amino acid sequence ILPRSPAF(SEQ ID NO: 250). In some embodiments, the CM comprises the amino acidsequence VAGRSMRP (SEQ ID NO: 251). In some embodiments, the CMcomprises the amino acid sequence VVPEGRRS (SEQ ID NO: 252). In someembodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ IDNO: 253). In some embodiments, the CM comprises the amino acid sequenceVLSKQMSF (SEQ ID NO: 254). In some embodiments, the CM comprises theamino acid sequence LKGRSYYY (SEQ ID NO: 255). In some embodiments, theCM comprises the amino acid sequence KRMPVQFL (SEQ ID NO: 256). In someembodiments, the CM comprises the amino acid sequence PQHRIVSF (SEQ IDNO: 257). In some embodiments, the CM comprises the amino acid sequenceYKKFVGSL (SEQ ID NO: 258). In some embodiments, the CM comprises theamino acid sequence HMMQYARH (SEQ ID NO: 259). In some embodiments, theCM comprises the amino acid sequence IPFSWSRF (SEQ ID NO: 260). In someembodiments, the CM comprises the amino acid sequence LSQARWRK (SEQ IDNO: 261). In some embodiments, the CM comprises the amino acid sequenceDISHWRRS (SEQ ID NO: 262). In some embodiments, the CM comprises theamino acid sequence RKTVQHWW (SEQ ID NO: 263_). In some embodiments, theCM comprises the amino acid sequence RFYRNQFF (SEQ ID NO: 264). In someembodiments, the CM comprises the amino acid sequence RSLVFAPI (SEQ IDNO: 265). In some embodiments, the CM comprises the amino acid sequenceRSPSRLKC (SEQ ID NO: 266). In some embodiments, the CM comprises theamino acid sequence RKMPNITV (SEQ ID NO: 267).

In some embodiments, the motif sequence includes a core CM consensussequence shown in Tables 10A-10D. In some embodiments, the motifsequence includes a subgenus, i.e., a subset, of the core CM consensussequence shown in Tables 10A-10D.

In some embodiments, the motif sequence includes an expanded consensussequence based on one of the core CM consensus sequence shown in Tables10A-10D. In some embodiments, the expanded consensus sequence is aconsensus sequence shown in Tables 11A-11D.

In some embodiments, the CM comprises a core CM consensus 11 sequencecomprising the amino acid sequence LSGRSANH (SEQ ID NO: 307) or LSGRSGNH(SEQ ID NO: 308). In some embodiments, the CM comprises an expanded coreCM consensus 11 sequence comprising the amino acid sequence DRLSGRSANHKK(SEQ ID NO: 309), DRLSGRSDNHKK (SEQ ID NO: 310), or NTLSGRSGNHGS (SEQ IDNO: 311).

In some embodiments, the CM comprises the amino acid sequence LSGRSANH(SEQ ID NO: 307). In some embodiments, the CM comprises the amino acidsequence LSGRSGNH (SEQ ID NO: 308). In some embodiments, the CMcomprises the amino acid sequence DRLSGRSANHKK (SEQ ID NO: 309). In someembodiments, the CM comprises the amino acid sequence DRLSGRSDNHKK (SEQID NO: 310). In some embodiments, the CM comprises the amino acidsequence NTLSGRSGNHGS (SEQ ID NO: 311).

In some embodiments, the CM comprises the amino acid sequence LSGRSANH(SEQ ID NO: 307). In some embodiments, the CM comprises the amino acidsequence LNGRSDNH (SEQ ID NO: 313). In some embodiments, the CMcomprises the amino acid sequence LTGRSDRH (SEQ ID NO: 314). In someembodiments, the CM comprises a core CM consensus 12 sequence comprisingthe amino acid sequence LSGRSANH (SEQ ID NO: 307), LNGRSDNH (SEQ ID NO:313), and LTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM anexpanded core CM consensus 12 sequence comprising an amino acid sequenceselected from the group consisting of DRLSGRSANHKK (SEQ ID NO: 309),DRLSGRSDNHKK (SEQ ID NO: 310), GPLNGRSDNHKA (SEQ ID NO: 320),GPLNGRSDNHKK (SEQ ID NO: 321), GPLNGRSDNHKR (SEQ ID NO: 322),GPLNGRSDNHQA (SEQ ID NO: 323), GPLNGRSDNHQK (SEQ ID NO: 324),GPLNGRSDNHQR (SEQ ID NO: 325), GPLNGRSDNHRA (SEQ ID NO: 326),GPLNGRSDNHRK (SEQ ID NO: 327), GPLNGRSDNHRR (SEQ ID NO: 328),RPLNGRSDNHKA (SEQ ID NO: 329), RPLNGRSDNHKK (SEQ ID NO: 330),RPLNGRSDNHKR (SEQ ID NO: 331), RPLNGRSDNHQA (SEQ ID NO: 332),RPLNGRSDNHQK (SEQ ID NO: 333), RPLNGRSDNHQR (SEQ ID NO: 334),RPLNGRSDNHRA (SEQ ID NO: 335), RPLNGRSDNHRK (SEQ ID NO: 336),RPLNGRSDNHRR (SEQ ID NO: 337), GPLSGRSDNHKA (SEQ ID NO: 338),GPLSGRSDNHKK (SEQ ID NO: 339), GPLSGRSDNHKR (SEQ ID NO: 340),GPLSGRSDNHQA (SEQ ID NO: 341), GPLSGRSDNHQK (SEQ ID NO: 342),GPLSGRSDNHQR (SEQ ID NO: 343), GPLSGRSDNHRA (SEQ ID NO: 344),GPLSGRSDNHRK (SEQ ID NO: 345), GPLSGRSDNHRR (SEQ ID NO: 346),RPLSGRSDNHKA (SEQ ID NO: 347), RPLSGRSDNHKK (SEQ ID NO: 348),RPLSGRSDNHKR (SEQ ID NO: 349), RPLSGRSDNHQA (SEQ ID NO: 350),RPLSGRSDNHQK (SEQ ID NO: 351), RPLSGRSDNHQR (SEQ ID NO: 352),RPLSGRSDNHRA (SEQ ID NO: 353), RPLSGRSDNHRK (SEQ ID NO: 354),RPLSGRSDNHRR (SEQ ID NO: 355), and KGLTGRSDRHQA (SEQ ID NO: 356).

In some embodiments, the CM comprises the amino acid sequenceDRLSGRSANHKK (SEQ ID NO: 309). In some embodiments, the CM comprises theamino acid sequence DRLSGRSDNHKK (SEQ ID NO: 310). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHKA (SEQ ID NO: 320).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHKK (SEQ ID NO: 321). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHKR (SEQ ID NO: 322). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHQA (SEQ ID NO: 323).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHQK (SEQ ID NO: 324). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHQR (SEQ ID NO: 325). In some embodiments,the CM comprises the amino acid sequence GPLNGRSDNHRA (SEQ ID NO: 326).In some embodiments, the CM comprises the amino acid sequenceGPLNGRSDNHRK (SEQ ID NO: 327). In some embodiments, the CM comprises theamino acid sequence GPLNGRSDNHRR (SEQ ID NO: 328). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHKA (SEQ ID NO: 329).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHKK (SEQ ID NO: 330). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHKR (SEQ ID NO: 331). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHQA (SEQ ID NO: 332).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHQK (SEQ ID NO: 333). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHQR (SEQ ID NO: 334). In some embodiments,the CM comprises the amino acid sequence RPLNGRSDNHRA (SEQ ID NO: 335).In some embodiments, the CM comprises the amino acid sequenceRPLNGRSDNHRK (SEQ ID NO: 336). In some embodiments, the CM comprises theamino acid sequence RPLNGRSDNHRR (SEQ ID NO: 337). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 338).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHKK (SEQ ID NO: 339). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHKR (SEQ ID NO: 340). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHQA (SEQ ID NO: 341).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHQK (SEQ ID NO: 342). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHQR (SEQ ID NO: 343). In some embodiments,the CM comprises the amino acid sequence GPLSGRSDNHKA (SEQ ID NO: 344).In some embodiments, the CM comprises the amino acid sequenceGPLSGRSDNHKK (SEQ ID NO: 345). In some embodiments, the CM comprises theamino acid sequence GPLSGRSDNHKR (SEQ ID NO: 346). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHKA (SEQ ID NO: 347).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHKK (SEQ ID NO: 348). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHKR (SEQ ID NO: 349). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHQA (SEQ ID NO: 350).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHQK (SEQ ID NO: 351). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHQR (SEQ ID NO: 352). In some embodiments,the CM comprises the amino acid sequence RPLSGRSDNHRA (SEQ ID NO: 353).In some embodiments, the CM comprises the amino acid sequenceRPLSGRSDNHKK (SEQ ID NO: 354). In some embodiments, the CM comprises theamino acid sequence RPLSGRSDNHKR (SEQ ID NO: 355). In some embodiments,the CM comprises the amino acid sequence KGLTGRSDRHQA (SEQ ID NO: 356).

In some embodiments, the CM comprises a core CM consensus 13 sequencecomprising the amino acid sequence RIGRSDNH (SEQ ID NO: 357) or RLGRSDNN(SEQ ID NO: 358). In some embodiments, the CM comprises an expanded coreCM consensus 13 sequence comprising the amino acid sequence NHRIGRSDNHRR(SEQ ID NO: 359) or TLRLGRSDNNKN (SEQ ID NO: 360).

In some embodiments, the CM comprises the amino acid sequence RIGRSDNH(SEQ ID NO: 357). In some embodiments, the CM comprises the amino acidsequence RLGRSDNN (SEQ ID NO: 358). In some embodiments, the CMcomprises the amino acid sequence NHRIGRSDNHRR (SEQ ID NO: 359). In someembodiments, the CM comprises the amino acid sequence TLRLGRSDNNKN (SEQID NO: 360).

In some embodiments, the CM comprises a core CM consensus 14 sequencecomprising an amino acid sequence selected from the group consisting ofTSGRSANP (SEQ ID NO: 361), TSGRSGNP (SEQ ID NO: 362), LSGRSANP (SEQ IDNO: 363), and LSGRSGNP (SEQ ID NO: 364). In some embodiments, the CMcomprises an expanded core CM consensus 14 sequence comprising an aminoacid sequence selected from the group consisting of TSTSGRSANPRG (SEQ IDNO: 365), TSTSGRSGNPRG (SEQ ID NO: 366), TSLSGRSANPRG (SEQ ID NO: 367),and TSLSGRSGNPRG (SEQ ID NO: 368).

In some embodiments, the CM comprises the amino acid sequence TSGRSANP(SEQ ID NO: 361). In some embodiments, the CM comprises the amino acidsequence TSGRSGNP (SEQ ID NO: 362). In some embodiments, the CMcomprises the amino acid sequence LSGRSANP (SEQ ID NO: 363). In someembodiments, the CM comprises the amino acid sequence LSGRSGNP (SEQ IDNO: 364). In some embodiments. In some embodiments, the CM comprises theamino acid sequence the CM comprises the amino acid sequenceTSTSGRSANPRG (SEQ ID NO: 365). In some embodiments, the CM comprises theamino acid sequence TSTSGRSGNPRG (SEQ ID NO: 366). In some embodiments,the CM comprises the amino acid sequence TSLSGRSANPRG (SEQ ID NO: 367).In some embodiments, the CM comprises the amino acid sequence andTSLSGRSGNPRG (SEQ ID NO: 368).

In some embodiments, the CM comprises an amino acid sequence selectedfrom the group consisting of LSGRSENH (SEQ ID NO: 369), SIARSDNL (SEQ IDNO: 370), LSGRSVTQ (SEQ ID NO: 371), LSGRSGNH (SEQ ID NO: 308), LTGRSDRH(SEQ ID NO: 314), LYGRSENN (SEQ ID NO: 374), RLGRSDNN (SEQ ID NO: 375),TSGRSANP (SEQ ID NO: 376), NTLSGRSENHSG (SEQ ID NO: 377), PPSIARSDNLAN(SEQ ID NO: 378), TGLSGRSVTQTS (SEQ ID NO: 379), NTLSGRSGNHGS (SEQ IDNO: 311), KGLTGRSDRHQA (SEQ ID NO: 381), KNLYGRSENNGN (SEQ ID NO: 382),TLRLGRSDNNKN (SEQ ID NO: 383), and TSTSGRSANPRG (SEQ ID NO: 384).

In some embodiments, the CM comprises the amino acid sequence LSGRSENH(SEQ ID NO: 369). In some embodiments, the CM comprises the amino acidsequence SIARSDNL (SEQ ID NO: 370). In some embodiments, the CMcomprises the amino acid sequence LSGRSVTQ (SEQ ID NO: 371). In someembodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ IDNO: 308). In some embodiments, the CM comprises the amino acid sequenceLTGRSDRH (SEQ ID NO: 314). In some embodiments, the CM comprises theamino acid sequence LYGRSENN (SEQ ID NO: 374). In some embodiments, theCM comprises the amino acid sequence RLGRSDNN (SEQ ID NO: 375). In someembodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ IDNO: 376). In some embodiments, the CM comprises the amino acid sequenceNTLSGRSENHSG (SEQ ID NO: 377). In some embodiments, the CM comprises theamino acid sequence PPSIARSDNLAN (SEQ ID NO: 378). In some embodiments,the CM comprises the amino acid sequence TGLSGRSVTQTS (SEQ ID NO: 379).In some embodiments, the CM comprises the amino acid sequenceNTLSGRSGNHGS (SEQ ID NO: 311). In some embodiments, the CM comprises theamino acid sequence KGLTGRSDRHQA (SEQ ID NO: 381). In some embodiments,the CM comprises the amino acid sequence KNLYGRSENNGN (SEQ ID NO: 382).In some embodiments, the CM comprises the amino acid sequenceTLRLGRSDNNKN (SEQ ID NO: 383). In some embodiments, the CM comprises theamino acid sequence TSTSGRSANPRG (SEQ ID NO: 384).

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, at least one protease is selected from matriptaseand uPA, and at least one protease is selected from the group consistingof those shown in Table 7.

In some embodiments, the activatable antibody includes at least a firstCM and a second CM. In some embodiments, the first CM and the second CMare each polypeptides of no more than 15 amino acids long. In someembodiments, the first CM and the second CM in the activatable antibodyhave the structural arrangement from N-terminus to C-terminus as followsin the uncleaved state: MM-CM1-CM2-AB, AB-CM2-CM1-MM, MM-CM2-CM1-AB, orAB-CM1-CM2-MM. In some embodiments, the activatable antibody includes alinking peptide between MM and CM1. In some embodiments, the activatableantibody includes a linking peptide between CM1 and CM2. In someembodiments, the activatable antibody includes a linking peptide betweenCM2 and AB. In some embodiments, the activatable antibody includes alinking peptide between MM and CM1 and a linking peptide between CM2 andAB. In some embodiments, the activatable antibody includes a linkingpeptide between MM and CM1 and a linking peptide between CM1 and CM2. Insome embodiments, the activatable antibody includes a linking peptidebetween CM1 and CM2 and a linking peptide between CM2 and AB. In someembodiments, the activatable antibody includes a linking peptide betweenMM and CM1, a linking peptide between CM1 and CM2, and a linking peptidebetween CM2 and AB.

In some embodiments, the CM2 is selected for use with a specificprotease. In some embodiments, the CM2 is a substrate for at least oneprotease selected from the group consisting of a matrix metalloprotease(MMP), a neutrophil elastase, uPA, legumain, matriptase, thrombin, acysteine protease such as a cathepsin, ADAM17, BMP-1, HtrA1, and aTMPRSS such as TMPRSS3 or TMPRSS4.

In some embodiments, the CM2 is a substrate for a neutrophil elastase.In some embodiments, the CM2 is a substrate for uPA. In someembodiments, the CM2 is a substrate for legumain. In some embodiments,the CM2 is a substrate for matriptase. In some embodiments, the CM2 is asubstrate for thrombin. In some embodiments, the CM2 is a substrate fora cysteine protease. In some embodiments, the CM2 is a substrate for acathepsin. In some embodiments, the CM2 is a substrate for ADAM17. Insome embodiments, the CM2 is a substrate for BMP-1. In some embodiments,the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is asubstrate for a TMPRSS. In some embodiments, the CM2 is a substrate forTMPRSS3. In some embodiments, the CM2 is a substrate for TMPRSS4.

For example, suitable CM2 are cleaved by at least one protease andinclude the sequence TGRGPSWV (SEQ ID NO: 402); SARGPSRW (SEQ ID NO:403); TARGPSFK (SEQ ID NO: 404); TARGPSW (SEQ ID NO: 405); LSGRSDNH (SEQID NO: 406); GGWHTGRN (SEQ ID NO: 407); HTGRSGAL (SEQ ID NO: 408);PLTGRSGG (SEQ ID NO: 409); AARGPAIH (SEQ ID NO: 411); RGPAFNPM (SEQ IDNO: 412); SSRGPAYL (SEQ ID NO: 413); RGPATPIM (SEQ ID NO: 414); RGPA(SEQ ID NO: 415); GGQPSGMWGW (SEQ ID NO: 416); FPRPLGITGL (SEQ ID NO:417); VHMPLGFLGP (SEQ ID NO: 418); SPLTGRSG (SEQ ID NO: 419); SAGFSLPA(SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 420); SGGPLGVR (SEQ ID NO: 421);PLGL (SEQ ID NO: 422); GPRSFGL (SEQ ID NO: 423) and/or GPRSFG (SEQ IDNO: 424).

In some embodiments, the CM2 comprises the amino acid sequence TGRGPSWV(SEQ ID NO: 402). In some embodiments, the CM2 comprises the amino acidsequence SARGPSRW (SEQ ID NO: 403). In some embodiments, the CM2comprises the amino acid sequence TARGPSFK (SEQ ID NO: 404). In someembodiments, the CM2 comprises the amino acid sequence TARGPSW (SEQ IDNO: 405). In some embodiments, the CM2 comprises the amino acid sequenceLSGRSDNH (SEQ ID NO: 406). In some embodiments, the CM2 comprises theamino acid sequence GGWHTGRN (SEQ ID NO: 407). In some embodiments, theCM2 comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 408). In someembodiments, the CM2 comprises the amino acid sequence PLTGRSGG (SEQ IDNO: 409). In some embodiments, the CM2 comprises the amino acid sequenceAARGPAIH (SEQ ID NO: 411). In some embodiments, the CM2 comprises theamino acid sequence RGPAFNPM (SEQ ID NO: 412). In some embodiments, theCM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 413). In someembodiments, the CM2 comprises the amino acid sequence RGPATPIM (SEQ IDNO: 414). In some embodiments, the CM2 comprises the amino acid sequenceRGPA (SEQ ID NO: 415). In some embodiments, the CM2 comprises the aminoacid sequence GGQPSGMWGW (SEQ ID NO: 416). In some embodiments, the CM2comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 417). In someembodiments, the CM2 comprises the amino acid sequence VHMPLGFLGP (SEQID NO: 418). In some embodiments, the CM2 comprises the amino acidsequence SPLTGRSG (SEQ ID NO: 419). In some embodiments, the CM2comprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 420). In someembodiments, the CM2 comprises the amino acid sequence SGGPLGVR (SEQ IDNO: 421). In some embodiments, the CM2 comprises the amino acid sequencePLGL (SEQ ID NO: 422). In some embodiments, the CM2 comprises the aminoacid sequence GPRSFGL (SEQ ID NO: 423). In some embodiments, the CM2comprises the amino acid sequence GPRSFG (SEQ ID NO: 424).

In some embodiments, the CM2 is a substrate for at least one MMP. Insome embodiments, the CM2 is a substrate for at least one MMP listed inthe Table 7. In some embodiments, the CM2 is a substrate for MMP9. Insome embodiments, the CM2 is a substrate for MMP14. In some embodiments,the CM2 is a substrate for two or more MMPs. In some embodiments, theCM2 is a substrate for at least MMP9 or MMP14. In some embodiments, theCM2 is a substrate for two or more MMPs. In some embodiments, the CM2 isa substrate for at least MMP9 and MMP14.

In some embodiments, CM2 is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 425); QNQALRMA (SEQ ID NO: 426); AQNLLGMV(SEQ ID NO: 427); STFPFGMF (SEQ ID NO: 428); PVGYTSSL (SEQ ID NO: 429);DWLYWPGI (SEQ ID NO: 430); MIAPVAYR (SEQ ID NO: 431); RPSPMWAY (SEQ IDNO: 432); WATPRPMR (SEQ ID NO: 433); FRLLDWQW (SEQ ID NO: 434); LKAAPRWA(SEQ ID NO: 435); GPSHLVLT (SEQ ID NO: 436); LPGGLSPW (SEQ ID NO: 437);MGLFSEAG (SEQ ID NO: 438); SPLPLRVP (SEQ ID NO: 439); RMHLRSLG (SEQ IDNO: 440); LAAPLGLL (SEQ ID NO: 441); AVGLLAPP (SEQ ID NO: 442); LLAPSHRA(SEQ ID NO: 443), PAGLWLDP (SEQ ID NO: 444); and/or ISSGLSS (SEQ ID NO:445).

In some embodiments, the first cleaving agent and the second cleavingagent are the protease selected from matriptase and uPA, and the firstCM and the second CM are different substrates for the enzyme. In someembodiments, the first cleaving agent and the second cleaving agent aredifferent proteases, where at least one protease is selected frommatriptase and uPA. In some embodiments, the first cleaving agent andthe second cleaving agent are co-localized in the target tissue. In someembodiments, the first CM and the second CM are cleaved by at least onecleaving agent selected from matriptase and uPA in the target tissue.

In some embodiments, the activatable antibody is exposed to and cleavedby a protease selected from matriptase and uPA such that, in theactivated or cleaved state, the activated antibody includes a lightchain amino acid sequence that includes at least a portion of LP2 and/orCM sequence after the protease has cleaved the CM.

In some embodiments, the CM comprises the non-prime side of the proteasecleavage site; that is, the CM comprises at least the P1 and P2 aminoacids, and in some embodiments comprises the P1, P2 and P3 amino acidsand in some embodiments comprises the P1, P2, P3, and P4 amino acids. Insome embodiments, the CM comprises the non-prime side and the prime sideof the protease cleavage site. In some embodiments, the CM comprises thenon-prime side but lacks at least part of the prime side of the proteasecleavage site. In some embodiments, the CM comprises the non-prime sidebut lacks the prime side of the protease cleavage site. Such a CM can belinked directly or through a linker to an antibody or other molecule asdisclosed herein, such as, but not limited to, a detection moiety.

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or a fragment thereof. In someembodiments, the agent is conjugated to the AB via a linker. In someembodiments, the linker is a cleavable linker. In some embodiments, theagent is a microtubule inhibitor. In some embodiments, the agent is anucleic acid damaging agent, such as a DNA alkylator or DNAintercalator, or other DNA damaging agent. In some embodiments, thelinker is a cleavable linker. In some embodiments, the agent isconjugated to the AB via a linker that includes at least oneMMP-cleavable substrate sequence. In some embodiments, the agent is anagent selected from the group listed in Table 3. In some embodiments,the agent is a dolastatin. In some embodiments, the agent is anauristatin or derivative thereof. In some embodiments, the agent isauristatin E or a derivative thereof. In some embodiments, the agent ismonomethyl auristatin E (MMAE). In some embodiments, the agent ismonomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof. In some embodiments, the agent is apyrrolobenzodiazepine.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent.

In some embodiments, the conjugated antibody includes a detectablelabel. In some embodiments, the detectable label includes an imagingagent, a contrasting agent, an enzyme, a fluorescent label, achromophore, a dye, one or more metal ions, or a ligand-based label. Insome embodiments, the imaging agent comprises a radioisotope. In someembodiments, the radioisotope is indium or technetium. In someembodiments, the contrasting agent comprises iodine, gadolinium or ironoxide. In some embodiments, the enzyme comprises horseradish peroxidase,alkaline phosphatase, or β-galactosidase. In some embodiments, thefluorescent label comprises yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), green fluorescent protein (GFP), modified redfluorescent protein (mRFP), red fluorescent protein tdimer2 (RFPtdimer2), HCRED, or a europium derivative. In some embodiments, theluminescent label comprises an N-methylacrydium derivative. In someembodiments, the label comprises an Alexa Fluor® label, such as AlexFluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-basedlabel comprises biotin, avidin, streptavidin or one or more haptens.

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer is joineddirectly to the MM of the activatable antibody in the structuralarrangement from N-terminus to C-terminus of spacer-MM-CM-AB. An exampleof a spacer joined directly to the N-terminus of MM of the activatableantibody is QGQSGQ (SEQ ID NO: 446). In some embodiments, the spacerincludes at least the amino acid sequence QGQSGQ (SEQ ID NO: 446).

In some embodiments, the AB of the activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, the AB can beengineered to include one or more disulfide bonds.

In some embodiments, the serum half-life of the activatable antibody islonger than that of the corresponding antibody; e.g., the pK of theactivatable antibody is longer than that of the corresponding antibody.In some embodiments, the serum half-life of the activatable antibody issimilar to that of the corresponding antibody. In some embodiments, theserum half-life of the activatable antibody is at least 15 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 11 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 9 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 8 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 7 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 5 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 4 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 3 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least2 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 24 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 20 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 18 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least16 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 14 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 10 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least8 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 6 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 4 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 3 hours when administered to an organism.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody is monospecific. In some embodiments, theactivatable antibody and/or conjugated activatable antibody ismultispecific, e.g., by way of non-limiting example, bispecific ortrifunctional. In some embodiments, the activatable antibody and/orconjugated activatable antibody is formulated as part of apro-Bispecific T Cell Engager (BITE) molecule. In some embodiments, theactivatable antibody and/or conjugated activatable antibody isformulated as part of a pro-Chimeric Antigen Receptor (CAR) modified Tcell or other engineered receptor.

The disclosure also provides compositions and methods that include anactivatable antibody that includes an antibody or antibody fragment (AB)that specifically binds a given target, where the AB is coupled to amasking moiety (MM) that decreases the ability of the AB to bind itstarget. In some embodiments, the activatable antibody further includes acleavable moiety (CM) that is a substrate for at least one proteaseselected from matriptase and uPA. The compositions and methods providedherein enable the attachment of one or more agents to one or morecysteine residues in the AB without compromising the activity (e.g., themasking, activating or binding activity) of the activatable antibody. Insome embodiments, the compositions and methods provided herein enablethe attachment of one or more agents to one or more cysteine residues inthe AB without reducing or otherwise disturbing one or more disulfidebonds within the MM. The compositions and methods provided hereinproduce an activatable antibody that is conjugated to one or moreagents, e.g., any of a variety of therapeutic, diagnostic and/orprophylactic agents, for example, in some embodiments, without any ofthe agent(s) being conjugated to the MM of the activatable antibody. Thecompositions and methods provided herein produce conjugated activatableantibodies in which the MM retains the ability to effectively andefficiently mask the AB of the activatable antibody in an uncleavedstate. The compositions and methods provided herein produce conjugatedactivatable antibodies in which the activatable antibody is stillactivated, i.e., cleaved, in the presence of a protease, i.e.,matriptase and/or uPA, that can cleave the CM.

The activatable antibodies have at least one point of conjugation for anagent, but in the methods and compositions provided herein less than allpossible points of conjugation are available for conjugation to anagent. In some embodiments, the one or more points of conjugation aresulfur atoms involved in disulfide bonds. In some embodiments, the oneor more points of conjugation are sulfur atoms involved in interchaindisulfide bonds. In some embodiments, the one or more points ofconjugation are sulfur atoms involved in interchain sulfide bonds, butnot sulfur atoms involved in intrachain disulfide bonds. In someembodiments, the one or more points of conjugation are sulfur atoms ofcysteine or other amino acid residues containing a sulfur atom. Suchresidues may occur naturally in the antibody structure or may beincorporated into the antibody by site-directed mutagenesis, chemicalconversion, or mis-incorporation of non-natural amino acids.

Also provided are methods of preparing a conjugate of an activatableantibody having one or more interchain disulfide bonds in the AB and oneor more intrachain disulfide bonds in the MM, and a drug reactive withfree thiols is provided. The method generally includes partiallyreducing interchain disulfide bonds in the activatable antibody with areducing agent, such as, for example, TCEP; and conjugating the drugreactive with free thiols to the partially reduced activatable antibody.As used herein, the term partial reduction refers to situations where anactivatable antibody is contacted with a reducing agent and less thanall disulfide bonds, e.g., less than all possible sites of conjugationare reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10% or less than 5% of all possible sites of conjugation arereduced.

In some embodiments, a method of reducing and conjugating an agent,e.g., a drug, to an activatable antibody resulting in selectivity in theplacement of the agent is provided. The method generally includespartially reducing the activatable antibody with a reducing agent suchthat any conjugation sites in the masking moiety or other non-AB portionof the activatable antibody are not reduced, and conjugating the agentto interchain thiols in the AB. The conjugation site(s) are selected soas to allow desired placement of an agent to allow conjugation to occurat a desired site. The reducing agent is, for example, TCEP. Thereduction reaction conditions such as, for example, the ratio ofreducing agent to activatable antibody, the length of incubation, thetemperature during the incubation, the pH of the reducing reactionsolution, etc., are determined by identifying the conditions thatproduce a conjugated activatable antibody in which the MM retains theability to effectively and efficiently mask the AB of the activatableantibody in an uncleaved state. The ratio of reduction agent toactivatable antibody will vary depending on the activatable antibody. Insome embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

In some embodiments, a method of reducing interchain disulfide bonds inthe AB of an activatable antibody and conjugating an agent, e.g., athiol-containing agent such as a drug, to the resulting interchainthiols to selectively locate agent(s) on the AB is provided. The methodgenerally includes partially reducing the AB with a reducing agent toform at least two interchain thiols without forming all possibleinterchain thiols in the activatable antibody; and conjugating the agentto the interchain thiols of the partially reduced AB. For example, theAB of the activatable antibody is partially reduced for about 1 hour atabout 37° C. at a desired ratio of reducing agent:activatable antibody.In some embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, TCEP. In someembodiments, the reduced activatable antibody can be purified prior toconjugation, using for example, column chromatography, dialysis, ordiafiltration. In some embodiments, the reduced antibody is not purifiedafter partial reduction and prior to conjugation.

The disclosure also provides partially reduced activatable antibodies inwhich at least one interchain disulfide bond in the activatable antibodyhas been reduced with a reducing agent without disturbing any intrachaindisulfide bonds in the activatable antibody, wherein the activatableantibody includes an antibody or an antigen binding fragment thereof(AB) that specifically binds to the target, a masking moiety (MM) thatinhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, wherein the CM is a polypeptide that functions as a substratefor at least one protease selected from matriptase and uPA. In someembodiments, the MM is coupled to the AB via the CM. In someembodiments, one or more intrachain disulfide bond(s) of the activatableantibody is not disturbed by the reducing agent. In some embodiments,one or more intrachain disulfide bond(s) of the MM within theactivatable antibody is not disturbed by the reducing agent. In someembodiments, the activatable antibody in the uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

The disclosure also provides conjugated activatable antibodies thatinclude an activatable antibody linked to monomethyl auristatin D (MMAD)payload, wherein the activatable antibody includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to atarget, a masking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and cleavablemoiety (CM) coupled to the AB, and the CM is a polypeptide thatfunctions as a substrate for at least one MMP protease.

In some embodiments, the MMAD-conjugated activatable antibody can beconjugated using any of several methods for attaching agents to ABs: (a)attachment to the carbohydrate moieties of the AB, or (b) attachment tosulfhydryl groups of the AB, or (c) attachment to amino groups of theAB, or (d) attachment to carboxylate groups of the AB.

In some embodiments, the MMAD payload is conjugated to the AB via alinker. In some embodiments, the MMAD payload is conjugated to acysteine in the AB via a linker. In some embodiments, the MMAD payloadis conjugated to a lysine in the AB via a linker. In some embodiments,the MMAD payload is conjugated to another residue of the AB via alinker, such as those residues disclosed herein. In some embodiments,the linker is a thiol-containing linker. In some embodiments, the linkeris a cleavable linker. In some embodiments, the linker is anon-cleavable linker. In some embodiments, the linker is selected fromthe group consisting of the linkers shown in Tables 5 and 6. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide caproyl-valine-citrulline linker. In some embodiments,the activatable antibody and the MMAD payload are linked via a maleimidePEG-valine-citrulline linker. In some embodiments, the activatableantibody and the MMAD payload are linked via a maleimidecaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker. In some embodiments, the MMAD payload is conjugated to the ABusing the partial reduction and conjugation technology disclosed herein.

The disclosure also provides polypeptides and other larger moleculesthat include one or more of the matriptase-cleavable substrate sequencespresented herein and/or uPA-cleavable substrate sequences presentedherein. By way of non-limiting example, matriptase-cleavable substratesequences presented herein and/or uPA-cleavable substrate sequencespresented herein are useful in prodrug compositions and methods of usethereof. These matriptase-cleavable substrate sequences presented hereinand/or uPA-cleavable substrate sequences presented herein are alsouseful in probes and other detection agents and methods of use thereof.For example, the matriptase-cleavable substrate sequences presentedherein and/or uPA-cleavable substrate sequences presented herein can beused in conjunction with fluors and other quenchers to produce detectionagents, such as imaging agents and/or other diagnostic agents. Those ofordinary skill in the art will appreciate that the matriptase-cleavablesubstrate sequences presented herein and/or uPA-cleavable substratesequences presented herein are useful in any composition and/or methodin the art that would use a substrate that is cleavable by matriptaseand/or uPA.

In some embodiments, the matriptase and/or uPA substrates of thedisclosure are used in larger molecules, for example, isolatedpolypeptides that include at least one additional moiety (M) selectedfrom the group consisting of (i) at least one moiety that is locatedamino (N) terminally to the CM (M_(N)), i.e., at a location within thelarger molecule that is situated closer to the N-terminus of the largermolecule than the CM; (ii) at least one moiety that is located carboxyl(C) terminally to the CM (M_(C)), i.e., at a location within the largermolecule that is situated closer to the C-terminus of the largermolecule than the CM; and (iii) combinations thereof. In someembodiments, the larger molecule includes at least one M_(N) and atleast one M_(C).

By way of non-limiting examples, suitable M_(N) for use in the largermolecules of the disclosure include at least one of the following: amasking moiety, an antibody, a protein, a therapeutic agent, anantineoplastic agent, a toxic agent, a drug, a detectable moiety, adiagnostic agent, an affinity tag, and combinations thereof.

By way of non-limiting examples, suitable M_(C) for use in the largermolecules of the disclosure include at least one of the following: amasking moiety, an antibody, a protein, a therapeutic agent, anantineoplastic agent, a toxic agent, a drug, a detectable moiety, adiagnostic agent, an affinity tag, and combinations thereof.

The disclosure also provides an isolated nucleic acid molecule encodinga CM-containing molecule of the disclosure, e.g., a CM-containingpolypeptide such as, e.g., a CM-containing probe, an antibody and/or anactivatable antibody described herein, as well as vectors that includethese isolated nucleic acid sequences. The disclosure provides methodsof producing CM-containing polypeptide by culturing a cell underconditions that lead to expression of the CM-containing polypeptide,wherein the cell comprises such a vector. The disclosure providesmethods of producing an antibody and/or activatable antibody byculturing a cell under conditions that lead to expression of theantibody and/or activatable antibody, wherein the cell comprises such avector.

The disclosure provides a method of manufacturing a CM-containingpolypeptide of the disclosure that binds a given target by (a) culturinga cell comprising a nucleic acid construct that encodes theCM-containing polypeptide under conditions that lead to expression ofthe polypeptide, (i) wherein the polypeptide includes a cleavable moiety(CM), and (ii) wherein the CM is a polypeptide that functions as asubstrate for at least one protease selected from matriptase and uPA;and (b) recovering the polypeptide. These methods can also include thefurther step of (c) conjugating the recovered polypeptide to one or moreadditional agents.

The disclosure provides a method of manufacturing a conjugated antibodyof the disclosure that binds a given target by (a) culturing a cellcomprising a nucleic acid construct that encodes the antibody underconditions that lead to expression of the antibody, (i) wherein theantibody includes a cleavable moiety (CM), and (ii) wherein the CM is apolypeptide that functions as a substrate for at least one proteaseselected from matriptase and uPA; (b) recovering the antibody; and (c)conjugating the recovered antibody to one or more additional agents.

The disclosure also provides a method of manufacturing the activatableantibodies of the disclosure that bind in an activated state a giventarget by (a) culturing a cell comprising a nucleic acid construct thatencodes the activatable antibody under conditions that lead toexpression of the activatable antibody, wherein the activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM), and anantibody or an antigen binding fragment thereof (AB) that specificallybinds the target, (i) wherein the CM is a polypeptide that functions asa substrate for a protease selected from matriptase and uPA; and (ii)wherein the CM is positioned in the activatable antibody such that, inan uncleaved state, the MM interferes with specific binding of the AB tothe target and in a cleaved state the MM does not interfere or competewith specific binding of the AB to the target; and (b) recovering theactivatable antibody.

The disclosure also provides methods of producing non-polypeptideCM-containing molecules, including by way of non-limiting examples,prodrugs, non-peptide probes, etc. These non-polypeptide CM-containingmolecules can be made using any of a variety of art-recognizedtechniques, including standard chemical synthesis and/or conjugationmethods.

The disclosure provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise ameliorating atarget-related disease in a subject by administering a therapeuticallyeffective amount of a conjugated antibody, an activatable antibodyand/or a conjugated activatable antibody described herein to a subjectin need thereof.

The disclosure provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise amelioratinginflammation and/or an inflammatory disorder in a subject byadministering a therapeutically effective amount of a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody described herein to a subject in need thereof. The disclosurealso provides methods of preventing, delaying the progression of,treating, alleviating a symptom of, or otherwise ameliorating cancer ina subject by administering a therapeutically effective amount of aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody described herein to a subject in need thereof. Thedisclosure also provides methods of preventing, delaying the progressionof, treating, alleviating a symptom of, or otherwise ameliorating anautoimmune disease in a subject by administering a therapeuticallyeffective amount a conjugated antibody, an activatable antibody and/or aconjugated activatable antibody described herein to a subject in needthereof.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody used in any of the embodiments of these methods anduses can be administered at any stage of the disease. For example, sucha conjugated antibody, activatable antibody and/or conjugatedactivatable antibody can be administered to a patient suffering cancerof any stage, from early to metastatic. The terms subject and patientare used interchangeably herein.

In some embodiments, the subject is a mammal, such as a human, non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a human. Insome embodiments, the subject is a companion animal. In someembodiments, the subject is an animal in the care of a veterinarian.

The conjugated antibody, activatable antibody and/or conjugatedactivatable antibody and therapeutic formulations thereof areadministered to a subject suffering from or susceptible to a disease ordisorder associated with aberrant target expression and/or activity. Asubject suffering from or susceptible to a disease or disorderassociated with aberrant target expression and/or activity is identifiedusing any of a variety of methods known in the art. For example,subjects suffering from cancer or other neoplastic condition areidentified using any of a variety of clinical and/or laboratory testssuch as, physical examination and blood, urine and/or stool analysis toevaluate health status. For example, subjects suffering frominflammation and/or an inflammatory disorder are identified using any ofa variety of clinical and/or laboratory tests such as physicalexamination and/or bodily fluid analysis, e.g., blood, urine and/orstool analysis, to evaluate health status.

Administration of a conjugated antibody, an activatable antibody and/ora conjugated activatable antibody to a patient suffering from a diseaseor disorder associated with aberrant target expression and/or activityis considered successful if any of a variety of laboratory or clinicalobjectives is achieved. For example, administration of a conjugatedantibody, an activatable antibody and/or a conjugated activatableantibody to a patient suffering from a disease or disorder associatedwith aberrant target expression and/or activity is considered successfulif one or more of the symptoms associated with the disease or disorderis alleviated, reduced, inhibited or does not progress to a further,i.e., worse, state. Administration of a conjugated antibody, anactivatable antibody and/or a conjugated activatable antibody to apatient suffering from a disease or disorder associated with aberranttarget expression and/or activity is considered successful if thedisease or disorder enters remission or does not progress to a further,i.e., worse, state.

In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody is administered during and/orafter treatment in combination with one or more additional agents suchas, for example, an anti-inflammatory agent, an immunosuppressive agent,and/or a chemotherapeutic agent. In some embodiments, the conjugatedantibody, activatable antibody and/or conjugated activatable antibodyand the additional agent(s) are administered simultaneously. Forexample, the conjugated antibody, activatable antibody and/or conjugatedactivatable antibody and the additional agent(s) can be formulated in asingle composition or administered as two or more separate compositions.In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody and the additional agent(s) areadministered sequentially, or the antibody and/or conjugated antibodiesand the additional agent are administered at different times during atreatment regimen. For example, the antibody and/or conjugatedantibodies is administered prior to the administration of the additionalagent, the antibody and/or conjugated antibodies is administeredsubsequent to the administration of the additional agent, or theantibody and/or conjugated antibodies and the additional agent areadministered in an alternating fashion. As described herein, theantibody and/or conjugated antibodies and additional agent areadministered in single doses or in multiple doses.

In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody is administered during and/orafter treatment in combination with one or more additional agents suchas, by way of non-limiting example, an anti-inflammatory agent, animmunosuppressive agent, a chemotherapeutic agent, such as an alkylatingagent, an anti-metabolite, an anti-microtubule agent, a topoisomeraseinhibitor, a cytotoxic antibiotic, and/or any other nucleic aciddamaging agent. In some embodiments, the additional agent is a taxane,such as paclitaxel (e.g., Abraxane®). In some embodiments, theadditional agent is an anti-metabolite, such as gemcitabine. In someembodiments, the additional agent is an alkylating agent, such asplatinum-based chemotherapy, such as carboplatin or cisplatin. In someembodiments, the additional agent is a targeted agent, such as a kinaseinhibitor, e.g., sorafenib or erlotinib. In some embodiments, theadditional agent is a targeted agent, such as another antibody, e.g., amonoclonal antibody (e.g., bevacizumab), a bispecific antibody, or amultispecific antibody. In some embodiments, the additional agent is aproteosome inhibitor, such as bortezomib or carfilzomib. In someembodiments, the additional agent is an immune modulating agent, such aslenolidominde or IL-2. In some embodiments, the additional agent isradiation. In some embodiments, the additional agent is an agentconsidered standard of care by those skilled in the art. In someembodiments, the additional agent is a chemotherapeutic agent well knownto those skilled in the art.

In some embodiments, the additional agent is an antibody, anotherconjugated antibody, another activatable antibody and/or anotherconjugated activatable antibody. In some embodiments the additionalagent is an antibody, another conjugated antibody, another activatableantibody and/or another conjugated activatable antibody against the sametarget as the first conjugated antibody, activatable antibody and/or aconjugated activatable antibody. In some embodiments the additionalagent is an antibody, another conjugated antibody, another activatableantibody and/or another conjugated activatable antibody against a targetdifferent than the target of the first conjugated antibody, activatableantibody and/or a conjugated activatable antibody.

In some embodiments, the conjugated antibody, activatable antibodyand/or conjugated activatable antibody and the additional agent(s) areadministered simultaneously. For example, the conjugated antibody,activatable antibody and/or conjugated activatable antibody and theadditional agent(s) can be formulated in a single composition oradministered as two or more separate compositions. In some embodiments,the conjugated antibody, activatable antibody and/or conjugatedactivatable antibody and the additional agent(s) are administeredsequentially, or the antibody and/or conjugated antibodies and theadditional agent are administered at different times during a treatmentregimen. For example, the antibody and/or conjugated antibodies isadministered prior to the administration of the additional agent, theantibody and/or conjugated antibodies is administered subsequent to theadministration of the additional agent, or the antibody and/orconjugated antibodies and the additional agent are administered in analternating fashion. As described herein, the antibody and/or conjugatedantibodies and additional agent are in single doses or in multipledoses.

In some embodiments, the CM is linked or otherwise attached to anactivatable antibody that includes an antibody or antigen-bindingfragment thereof that specifically binds a given target coupled to amasking moiety (MM), such that coupling of the MM to the AB reduces theability of the antibody or antigen-binding fragment thereof to bind thetarget. In some embodiments, the MM is coupled via the CM. Exemplarytargets include, but are not limited to the targets shown in Table 1.Exemplary ABs include, but are not limited to, the targets shown inTable 2. The activatable antibodies provided herein are stable incirculation, activated at intended sites of therapy and/or diagnosis butnot in normal, e.g., healthy tissue or other tissue not targeted fortreatment and/or diagnosis, and, when activated, exhibit binding to thetarget that is at least comparable to the corresponding, unmodifiedantibody.

The disclosure also provides methods and kits for using the conjugatedantibodies, activatable antibodies and/or conjugated activatableantibodies in a variety of diagnostic and/or prophylactic indications.

In some embodiments, the disclosure provides methods and kits fordetecting presence or absence of a cleaving agent and a target ofinterest in a subject or a sample by (i) contacting a subject or samplewith an activatable antibody, wherein the activatable antibody comprisesa masking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the target of interest, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of theAB to the target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the target, and in a cleaved, activated state theMM does not interfere or compete with specific binding of the AB to thetarget; and (ii) measuring a level of activated activatable antibody inthe subject or sample, wherein a detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent and the target are present in the subject or sample andwherein no detectable level of activated activatable antibody in thesubject or sample indicates that the cleaving agent, the target or boththe cleaving agent and the target are absent in the subject or sample.

In some embodiments, the activatable antibody is an activatable antibodyto which a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the activatable antibody comprises a detectable label. In someembodiments, the detectable label is positioned on the AB. In someembodiments, measuring the level of activatable antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

In some embodiments of these methods and kits, the activatable antibodyincludes a detectable label. In some embodiments of these methods andkits, the detectable label includes an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, or a ligand-based label. In some embodiments of thesemethods and kits, the imaging agent comprises a radioisotope. In someembodiments of these methods and kits, the radioisotope is indium ortechnetium. In some embodiments of these methods and kits, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods and kits, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or β-galactosidase. In someembodiments of these methods and kits, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods and kits, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods and kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods, the subject is a human. In someembodiments, the subject is a non-human mammal, such as a non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a rodent.

In some embodiments of these methods and kits, the method is an in vivomethod. In some embodiments of these methods, the method is an in situmethod. In some embodiments of these methods, the method is an ex vivomethod. In some embodiments of these methods, the method is an in vitromethod.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with a probe comprising a cleavable moiety (CM) and adetectable label that is released or activated following cleavage of theCM; and (ii) measuring a level of detectable label in the subject orbiological sample. When such release or activation increases detectionof the label (e.g., stimulates a detectable signal), a detectable levelof the detectable label in the subject or biological sample indicatesthat the cleaving agent is present in the subject or biological sample,and wherein a reduced detectable level of the detectable label in thesubject or biological sample indicates that the cleaving agent is absentand/or not sufficiently present in the subject or biological sample at adetectable level, such that protease cleavage of the CM cannot bedetected in the subject or biological sample. When such release oractivation reduces detection of the label, a detectable level of thedetectable label in the subject or biological sample indicates that thecleaving agent is absent and/or not sufficiently present in the subjector biological sample at a detectable level, such that protease cleavageof the CM cannot be detected in the subject or biological sample, andwherein a reduced detectable level of the detectable label in thesubject or biological sample indicates that the cleaving agent ispresent in the subject or biological sample.

In some embodiments of these methods and kits, the probes comprising CMincludes a detectable label. In some embodiments of these methods andkits, the detectable label includes an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, or a ligand-based label. In some embodiments of thesemethods and kits, the imaging agent comprises a radioisotope. In someembodiments of these methods and kits, the radioisotope is indium ortechnetium. In some embodiments of these methods and kits, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods and kits, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or β-galactosidase. In someembodiments of these methods and kits, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods and kits, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods and kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods and kits, the method is used toidentify or otherwise refine a patient population suitable for treatmentwith an activatable antibody of the disclosure, followed by treatment byadministering that activatable antibody and/or conjugated activatableantibody to a subject in need thereof. For example, patients that testpositive for both the target and at least one protease selected frommatriptase and uPA that cleaves the substrate in the cleavable moiety(CM) of the activatable antibody being tested in these methods areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM, and the patient is then administered atherapeutically effective amount of the activatable antibody and/orconjugated activatable antibody that was tested. Likewise, patients thattest negative for either or both of the target and the protease, i.e.,matriptase and/or uPA that cleaves the substrate in the CM in theactivatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other activatableantibodies until a suitable activatable antibody for treatment isidentified (e.g., an activatable antibody comprising a CM that iscleaved by the patient at the site of disease). In some embodiments, thepatient is then administered a therapeutically effective amount of theactivatable antibody and/or conjugated for which the patient testedpositive.

The disclosure also provides polypeptides and other larger moleculesthat include one or more of the matriptase-cleavable substrate sequencespresented herein and/or uPA-cleavable substrate sequences presentedherein. By way of non-limiting example, matriptase-cleavable substratesequences presented herein and/or uPA-cleavable substrate sequencespresented herein are useful in prodrug compositions and methods of usethereof. In some embodiments, the polypeptide comprises a CM joined to adrug, such as a small molecule. Examples of drugs are well known in theart. These matriptase-cleavable substrate sequences presented hereinand/or uPA-cleavable substrate sequences presented herein are alsouseful in probes and other detection agents and methods of use thereof.For example, the matriptase-cleavable substrate sequences presentedherein and/or uPA-cleavable substrate sequences presented herein can beused in conjunction with fluors and other quenchers to produce detectionagents, such as imaging agents and/or other diagnostic agents. Those ofordinary skill in the art will appreciate that the matriptase-cleavablesubstrate sequences presented herein and/or uPA-cleavable substratesequences presented herein are useful in any composition and/or methodin the art that would use a substrate that is cleavable by matriptaseand/or uPA.

In some embodiments, the matriptase and/or uPA substrates of thedisclosure are used in larger molecules, for example, isolatedpolypeptides that include at least one additional moiety (M) selectedfrom the group consisting of (i) at least one moiety that is locatedamino (N) terminally to the CM (M_(N)), i.e., at a location within thelarger molecule that is situated closer to the N-terminus of the largermolecule than the CM; (ii) at least one moiety that is located carboxyl(C) terminally to the CM (M_(C)), i.e., at a location within the largermolecule that is situated closer to the C-terminus of the largermolecule than the CM; and (iii) combinations thereof. In someembodiments, the larger molecule includes at least one M_(N) and atleast one M_(C).

By way of non-limiting examples, suitable M_(N) for use in the largermolecules of the disclosure include at least one of the following: amasking moiety, an antibody, a protein, a therapeutic agent, anantineoplastic agent, a toxic agent, a drug, a detectable moiety, adiagnostic agent, an affinity tag, and combinations thereof.

By way of non-limiting examples, suitable M_(C) for use in the largermolecules of the disclosure include at least one of the following: amasking moiety, an antibody, a protein, a therapeutic agent, anantineoplastic agent, a toxic agent, a drug, a detectable moiety, adiagnostic agent, an affinity tag, and combinations thereof.

Pharmaceutical compositions according to the disclosure can include anantibody of the disclosure and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs depicting cleavage of pool SMP30 bymatriptase-1.

FIG. 2 is a series of graphs depicting cleavage of pool SMP17 bymatriptase-1 and resistance to cleavage by tPA.

FIG. 3 is a series of graphs depicting cleavage of the substratesequence VAGRSMRP (SEQ ID NO: 251) by matriptase-1.

FIGS. 4A and 4B are a series of schematic representations of the peptidedisplay platforms used in the working examples provided herein. FIG. 4Ais a schematic representation of the sequence of the display platformreferred to herein as “Display Platform CYTX-DP-XXXXXXXX” or“CYTX-DP-XXXXXXXX” (SEQ ID NO: 694). FIG. 4B is a schematicrepresentation of the sequence of the display platform referred toherein as “Display Platform SP-CYTX-DP-XXXXXXXX” or“SP-CYTX-DP-XXXXXXXX” (SEQ ID NO: 695), where SP-CYTX-DP-XXXXXXXX is theCYTX-DP-XXXXXXXX platform with a signal peptide.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides amino acid sequences that include a cleavablemoiety (CM) that is a substrate for at least one protease selected frommatriptase and u-plasminogen activator (uPA). These CMs are useful in avariety of therapeutic, diagnostic and prophylactic indications.

The disclosure provides antibodies that include one or more of thesematriptase-cleavable substrates and/or uPA-cleavable substrates. Forexample, these matriptase-cleavable substrates and/or uPA-cleavablesubstrates are useful when conjugating antibodies to one or moreadditional agents to produce conjugated antibodies. Thesematriptase-cleavable substrates and/or uPA-cleavable substrates areuseful in activatable antibody constructs.

The conjugated antibodies include an antibody or antigen-bindingfragment thereof that specifically binds a target, and the activatableantibodies include an antibody or antigen-binding fragment thereof (AB)that specifically binds a target. Exemplary classes of targets of anantibody or antigen-binding fragment thereof include, but are notnecessarily limited to, cell surface receptors and secreted bindingproteins (e.g., growth factors), soluble enzymes, structural proteins(e.g. collagen, fibronectin) and the like. In some embodiments,conjugated antibodies and/or activatable antibodies have an antibody orantigen-binding fragment thereof that binds an extracellular target,usually an extracellular protein target. In some embodiments, conjugatedantibodies and/or activatable antibodies are designed for cellularuptake and are switchable inside a cell.

As a non-limiting example, the antibody or antigen-binding fragmentand/or the AB of an activatable antibody is a binding partner for anytarget listed in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 Alpha-4 integrin Alpha-V integrinalpha4beta1 integrin alpha4beta7 integrin AGR2 Anti-Lewis-Y Apelin Jreceptor APRIL B7-H4 BAFF BTLA C5 complement C-242 CA9 CA19-9 (Lewis a)Carbonic anhydrase 9 CD2 CD3 CD6 CD9 CD11a CD19 CD20 CD22 CD24 CD25 CD27CD28 CD30 CD33 CD38 CD40 CD40L CD41 CD44 CD44v6 CD47 CD51 CD52 CD56 CD64CD70 CD71 CD74 CD80 CD81 CD86 CD95 CD117 CD125 CD132 (IL-2RG) CD133CD137 CD138 CD166 CD172A CD248 CDH6 CEACAM5 (CEA) CEACAM6 (NCA-90)CLAUDIN-3 CLAUDIN-4 cMet Collagen Cripto CSFR CSFR-1 CTLA-4 CTGF CXCL10CXCL13 CXCR1 CXCR2 CXCR4 CYR61 DL44 DLK1 DLL4 DPP-4 DSG1 EGFR EGFRviiiEndothelin B receptor (ETBR) ENPP3 EpCAM EPHA2 EPHB2 ERBB3 F protein ofRSV FAP FGF-2 FGF8 FGFR1 FGFR2 FGFR3 FGFR4 Folate receptor GAL3ST1 G-CSFG-CSFR GD2 GITR GLUT1 GLUT4 GM-CSF GM-CSFR GP IIb/IIIa receptors Gp130GPIIB/IIIA GPNMB GRP78 HER2/neu HGF hGH HVEM Hyaluronidase ICOS IFNalphaIFNbeta IFNgamma IgE IgE Receptor (FceRI) IGF IGF1R IL1B IL1R IL2 IL11IL12 IL12p40 IL-12R, IL-12Rbeta1 IL13 IL13R IL15 IL17 IL18 IL21 IL23IL23R IL27/IL27R (wsx1) IL29 IL-31R IL31/IL31R IL2R IL4 IL4R IL6, IL6RInsulin Receptor Jagged Ligands Jagged 1 Jagged 2 LAG-3 LIF-R Lewis XLIGHT LRP4 LRRC26 MCSP Mesothelin MRP4 MUC1 Mucin-16 (MUC16, CA-125)Na/K ATPase Neutrophil elastase NGF Nicastrin Notch Receptors Notch 1Notch 2 Notch 3 Notch 4 NOV OSM-R OX-40 PAR2 PDGF-AA PDGF-BB PDGFRalphaPDGFRbeta PD-1 PD-L1 PD-L2 Phosphatidyl- serine P1GF PSCA PSMA RAAG12RAGE SLC44A4 Sphingosine 1 Phosphate STEAP1 STEAP2 TAG-72 TAPA1 TGFbetaTIGIT TIM-3 TLR2 TLR4 TLR6 TLR7 TLR8 TLR9 TMEM31 TNFalpha TNFR TNFRS12ATRAIL-R1 TRAIL-R2 Transferrin Transferrin receptor TRK-A TRK-B uPAR VAP1VCAM-1 VEGF VEGF-A VEGF-B VEGF-C VEGF-D VEGFR1 VEGFR2 VEGFR3 VISTAWISP-1 WISP-2 WISP-3

As a non-limiting example, the antibody or antigen-binding fragmentand/or the AB of an activatable antibody is or is derived from anantibody listed in Table 2.

TABLE 2 Exemplary sources for Abs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20Ocrelizumab CD20 Arzerra ™ (ofatumumab) CD20 Obinutuzumab CD20 Zenapax ™(daclizumab) CD25 Adcetris ™ (brentuximab vedotin) CD30 Myelotarg ™(gemtuzumab) CD33 Mylotarg ™ (gemtuzumab ozogamicin) CD33 Campath ™(alemtuzumab) CD52 ReoPro ™ (abiciximab) Glycoprotein receptor IIb/IIIaXolair ™ (omalizumab) IgE Herceptin ™ (trastuzumab) Her2 Kadcyla ™(trastuzumab emtansine) Her2 Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab)Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4(tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR(mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3Antril ™, Kineret ™ (ankinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R(tocilizumab) IL-6 receptor Stelara ™ (ustekinumab) IL-12/IL-23 Prolia ™(denosumab) RANKL

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the antibody referred to herein as the“Av1”antibody, which binds interleukin-6 receptor (IL-6R). The amino acidsequences for the Av1 heavy chain and the Av1 light chain are shownbelow in SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

Av1 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 447)QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Av1 Antibody LightChain Amino Acid Sequence: (SEQ ID NO: 448)DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the Av1 antibody and a masking moiety.Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include an amino acid sequence attached to the N-terminus ofthe AV1 light chain. These N-terminal amino acid sequences include, forexample, YGSCSWNYVHIFMDC (SEQ ID NO: 449); QGDFDIPFPAHWVPIT (SEQ ID NO:450); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 451); QGQSGQYGSCSWNYVHIFMDC (SEQ IDNO: 452); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 453); orQGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 454). It is also to be appreciatedthat such amino acid sequences can be attached to the N-terminus of theAV1 heavy chain or to the C-terminus of the AV1 heavy or light chain.

Exemplary activatable antibodies of the disclosure include, for example,antibodies that bind Epidermal Growth Factor Receptor (EGFR) and thatinclude a heavy chain and a light chain that are, or are derived from,an antibody selected from the group consisting of the antibody referredto herein as the“c225v5” antibody, the antibody referred to herein asthe“c225v4” antibody, and the antibody referred to herein as the“c225v6”antibody, each of which binds EGFR. The c225v5 antibody, the c225v4antibody, and the c225v6 antibody share the same light chain sequence,referred to herein as “c225 light chain.” The amino acid sequences forthe c225v5 heavy chain, the c225v4 antibody, the c225v6 antibody, andthe c225 light chain are shown below.

C225v5 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 455)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* C225v4 Antibody HeavyChain Amino Acid Sequence: (SEQ ID NO: 456)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* C225v6 Antibody HeavyChain Amino Acid Sequence: (SEQ ID NO: 457)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* C225 Antibody LightChain Amino Acid Sequence: (SEQ ID NO: 458)QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC*

Exemplary activatable antibodies of the disclosure include, for example,antibodies that bind EGFR, that include a heavy chain and a light chainthat are, or are derived from, the c225v5 antibody, and that include amasking moiety, a first linking peptide, a cleavable moiety, and asecond linking peptide. In some embodiments, the heavy chain and/or thelight chain includes a signal peptide. The heavy chain and light chainamino acid sequences for c225v5 without the signal peptide are shownabove in SEQ ID NO: 455 (heavy chain without signal peptide) and SEQ IDNO: 458 (light chain without signal peptide). In some embodiments, theactivatable anti-EGFR antibody includes a combination of the amino acidsequences shown in SEQ ID NO: 455, SEQ ID NO: 458 and/or the nucleicacid and amino acid sequences shown below:

C225v5 Antibody Heavy Chain Nucleic Acid Sequence with Signal Peptide:(SEQ ID NO: 684) ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGAUnderlined: Signal peptide C225v5 Antibody Heavy Chain Amino AcidSequence with Signal Peptide: (SEQ ID NO: 685)MYRMQLLSCIALSLALVTNSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK*Underlined: Signal peptide 3954-2787-c225 Light Chain Nucleic AcidSequence with Signal Peptide: (SEQ ID NO: 686)ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGCAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTACCTCCACCTCCGGCCGTTCCGCGAACCCGCGTGGTGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG Underlined:Signal Peptide 3954-2787-c225 Light Chain Amino Acid Sequence withSignal Peptide: (SEQ ID NO: 687)MYRMQLLSCIALSLALVTNSQGQSGQCISPRGCPDGPYVMYGSSGGSGGSGGSGTSTSGRSANPRGGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Underlined: Signal Peptide3954-2787-c225 Light Chain Nucleic Acid Sequence (without SignalPeptide): (SEQ ID NO: 688)CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTACCTCCACCTCCGGCCGTTCCGCGAACCCGCGTGGTGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 3954-2787-c225 LightChain Amino Acid Sequence (without Signal Peptide): (SEQ ID NO: 689)QGQSGQCISPRGCPDGPYVMYGSSGGSGGSGGSGTSTSGRSANPRGGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC*

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind a Jagged target,e.g., Jagged-1, Jagged-2 and/or both Jagged-1 and Jagged-2, and thatinclude a combination of a variable heavy chain region and a variablelight chain region that are, or are derived from, the variable heavychain and variable light chain sequences shown below.

Variable Light Chain Amino Sequence Lc4 (SEQ ID NO: 459)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc4 (SEQ ID NO: 460)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc5 (SEQ ID NO: 461)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc5 (SEQ ID NO: 462)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PYHGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc7 (SEQ ID NO: 463)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc7 (SEQ ID NO: 464)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc8 (SEQ ID NO: 465)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc8 (SEQ ID NO: 466)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHI GRTNPFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc13 (SEQ ID NO: 467)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc13 (SEQ ID NO: 468)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc16 (SEQ ID NO: 469)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc16 (SEQ ID NO: 470)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PYYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc19 (SEQ ID NO: 471)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc19 (SEQ ID NO: 472)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc21 (SEQ ID NO: 473)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc21 (SEQ ID NO: 474)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc24 (SEQ ID NO: 475)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc24 (SEQ ID NO: 476)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc26 (SEQ ID NO: 477)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc26 (SEQ ID NO: 478)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc27 (SEQ ID NO: 479)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc27 (SEQ ID NO: 480)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc28 (SEQ ID NO: 481)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc28 (SEQ ID NO: 482)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc30 (SEQ ID NO: 483)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc30 (SEQ ID NO: 484)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAA AFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc31 (SEQ ID NO: 485)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc31 (SEQ ID NO: 486)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc32 (SEQ ID NO: 487)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc32 (SEQ ID NO: 488)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc37 (SEQ ID NO: 489)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc37 (SEQ ID NO: 490)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PHNGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc39 (SEQ ID NO: 491)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc39 (SEQ ID NO: 492)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc40 (SEQ ID NO: 493)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR Heavy ChainAmino Sequence Hc40 (SEQ ID NO: 494)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc47 (SEQ ID NO: 495)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR VariableHeavy Chain Amino Sequence Hc47 (SEQ ID NO: 496)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable 4B2 Light Chain (SEQ ID NO: 497)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQ GTKVEIKR Variable 4B2Heavy Chain (SEQ ID NO: 498)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 4D11 Light Chain (SEQ ID NO: 499)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ GTKVEIKR Variable4D11 Heavy Chain (SEQ ID NO: 500)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 4E7 Light Chain (SEQ ID NO: 501)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQ GTKVEIKR Variable 4E7Heavy Chain (SEQ ID NO: 502)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable 4E11 Light Chain (SEQ ID NO: 503)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQ GTKVEIKR Variable4E11 Heavy Chain (SEQ ID NO: 504)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 6B7 Light Chain (SEQ ID NO: 505)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQ GTKVEIKR Variable 6B7Heavy Chain (SEQ ID NO: 506)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable 6F8 Light Chain (SEQ ID NO: 507)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQ GTKVEIKR Variable 6F8Heavy Chain (SEQ ID NO: 508)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS

Exemplary conjugated antibodies and/or activatable antibodies of thedisclosure include, for example, antibodies that bind a Jagged target,e.g., Jagged-1, Jagged-2 and/or both Jagged-1 and Jagged-2, and thatinclude a combination of a heavy chain region and a light chain regionthat are, or are derived from, the heavy chain and light chain sequencesshown below.

4D11 Light Chain sequence: (SEQ ID NO: 509)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 4D11Heavy Chain sequence: (SEQ ID NO: 510)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Heavy Chainsequence (SEQ ID NO: 511)EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Light ChainSequence (SEQ ID NO: 512)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The activatable antibodies provided herein contain at least an antibodyor antibody fragment thereof (collectively referred to as AB throughoutthe disclosure) that specifically binds a target, e.g., a human target,wherein the AB is modified by a masking moiety (MM).

In some embodiments, the masking moiety is selected for use with aspecific antibody or antibody fragment. For example, suitable maskingmoieties for use with antibodies that bind EGFR include MMs that includethe sequence CISPRG (SEQ ID NO: 513). By way of non-limiting examples,the MM can include a sequence such as CISPRGC (SEQ ID NO: 690) CISPRGCG(SEQ ID NO: 514); CISPRGCPDGPYVMY (SEQ ID NO: 515); CISPRGCPDGPYVM (SEQID NO: 516), CISPRGCEPGTYVPT (SEQ ID NO: 517) and CISPRGCPGQIWHPP (SEQID NO: 518). Other suitable masking moieties include any of theEGFR-specific masks disclosed in PCT Publication No. WO 2010/081173,such as, by way of non-limiting example, GSHCLIPINMGAPSC (SEQ ID NO:519); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 520);CNHHYFYTCGCISPRGCPG (SEQ ID NO: 521); ADHVFWGSYGCISPRGCPG (SEQ ID NO:522); CHHVYWGHCGCISPRGCPG (SEQ ID NO: 523); CPHFTTTSCGCISPRGCPG (SEQ IDNO: 524); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 525); CPHVSFGSCGCISPRGCPG (SEQID NO: 526); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 527); CNHVYFGTCGCISPRGCPG(SEQ ID NO: 528); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 529);CHHFTLTTCGCISPRGCPG (SEQ ID NO: 530); YNPCATPMCCISPRGCPG (SEQ ID NO:531); CNHHYFYTCGCISPRGCG (SEQ ID NO: 532); CNHHYHYYCGCISPRGCG (SEQ IDNO: 533); CNHVYFGTCGCISPRGCG (SEQ ID NO: 534); CHHVYWGHCGCISPRGCG (SEQID NO: 535); CPHFTTTSCGCISPRGCG (SEQ ID NO: 536); CNHFTLTTCGCISPRGCG(SEQ ID NO: 537); CHHFTLTTCGCISPRGCG (SEQ ID NO: 538);CPYYTLSYCGCISPRGCG (SEQ ID NO: 539); CPHVSFGSCGCISPRGCG (SEQ ID NO:540); ADHVFWGSYGCISPRGCG (SEQ ID NO: 541); YNPCATPMCCISPRGCG (SEQ ID NO:542); CHHVYWGHCGCISPRGCG (SEQ ID NO: 543);C(N/P)H(H/V/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO:544); CISPRGCGQPIPSVK (SEQ ID NO: 545); CISPRGCTQPYHVSR (SEQ ID NO:546); and/or CISPRGCNAVSGLGS (SEQ ID NO: 547).

Suitable masking moieties for use with antibodies that bind a Jaggedtarget, e.g., Jagged 1 and/or Jagged 2, include, by way of non-limitingexample, masking moieties that include a sequence such asQGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 691); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQID NO: 548); PWCMQRQDFLRCPQP (SEQ ID NO: 549); QLGLPAYMCTFECLR (SEQ IDNO: 550); CNLWVSGGDCGGLQG (SEQ ID NO: 551); SCSLWTSGSCLPHSP (SEQ ID NO:552); YCLQLPHYMQAMCGR (SEQ ID NO: 553); CFLYSCTDVSYWNNT (SEQ ID NO:554); PWCMQRQDYLRCPQP (SEQ ID NO: 555); CNLWISGGDCRGLAG (SEQ ID NO:556); CNLWVSGGDCRGVQG (SEQ ID NO: 557); CNLWVSGGDCRGLRG (SEQ ID NO:558); CNLWISGGDCRGLPG (SEQ ID NO: 559); CNLWVSGGDCRDAPW (SEQ ID NO:560); CNLWVSGGDCRDLLG (SEQ ID NO: 561); CNLWVSGGDCRGLQG (SEQ ID NO:562); CNLWLHGGDCRGWQG (SEQ ID NO: 563); CNIWLVGGDCRGWQG (SEQ ID NO:564); CTTWFCGGDCGVMRG (SEQ ID NO: 565); CNIWGPSVDCGALLG (SEQ ID NO:566); CNIWVNGGDCRSFEG (SEQ ID NO: 567); YCLNLPRYMQDMCWA (SEQ ID NO:568); YCLALPHYMQADCAR (SEQ ID NO: 569); CFLYSCGDVSYWGSA (SEQ ID NO:570); CYLYSCTDSAFWNNR (SEQ ID NO: 571); CYLYSCNDVSYWSNT (SEQ ID NO:572); CFLYSCTDVSYW (SEQ ID NO: 573); CFLYSCTDVAYWNSA (SEQ ID NO: 574);CFLYSCTDVSYWGDT (SEQ ID NO: 575); CFLYSCTDVSYWGNS (SEQ ID NO: 576);CFLYSCTDVAYWNNT (SEQ ID NO: 577); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 578);CFLYSCTDVAYWSGL (SEQ ID NO: 579); CYLYSCTDGSYWNST (SEQ ID NO: 580);CFLYSCSDVSYWGNI (SEQ ID NO: 581); CFLYSCTDVAYW (SEQ ID NO: 582);CFLYSCTDVSYWGST (SEQ ID NO: 583); CFLYSCTDVAYWGDT (SEQ ID NO: 584);GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 585); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO:586); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 587); GCNIWANGGDCRGWIDNIDG (SEQID NO: 588); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 589); GCNIWLVGGDCRGWLEEAVG(SEQ ID NO: 590); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 591);GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 592); GQQQWCNIWINGGDCRGWNG (SEQ ID NO:593); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 594); GTPGGCNIWANGGDCRGWEG (SEQID NO: 595); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 596); GCNIWLVGGDCRPWVEGG(SEQ ID NO: 597); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 598);GCNIWLNGGDCRAWVDTG (SEQ ID NO: 599); GCNIWIVGGDCRPFINDG (SEQ ID NO:600); GCNIWLNGGDCRPVVFGG (SEQ ID NO: 601); GCNIWLSGGDCRMFMNEG (SEQ IDNO: 602); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 603); GCNIWLNGGDCRGWEASG (SEQID NO: 604); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 605); GCNIWLNGGDCRTFVASG(SEQ ID NO: 606); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 607);GFLENCNIWLNGGDCRTG (SEQ ID NO: 608); GIYENCNIWLNGGDCRMG (SEQ ID NO:609); and/or GIPDNCNIWINGGDCRYG (SEQ ID NO: 610).

Suitable masking moieties for use with antibodies that bind aninterleukin 6 target, e.g., interleukin 6 receptor (IL-6R), include, byway of non-limiting example, masking moieties that include a sequencesuch as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 611); QGQSGQGDFDIPFPAHWVPIT(SEQ ID NO: 612); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 613);YRSCNWNYVSIFLDC (SEQ ID NO: 614); PGAFDIPFPAHWVPNT (SEQ ID NO: 615);ESSCVWNYVHIYMDC (SEQ ID NO: 616); YPGCKWNYDRIFLDC (SEQ ID NO: 617);YRTCSWNYVGIFLDC (SEQ ID NO: 618); YGSCSWNYVHIFMDC (SEQ ID NO: 619);YGSCSWNYVHIFLDC (SEQ ID NO: 620); YGSCNWNYVHIFLDC (SEQ ID NO: 621);YTSCNWNYVHIFMDC (SEQ ID NO: 622); YPGCKWNYDRIFLDC (SEQ ID NO: 623);WRSCNWNYAHIFLDC (SEQ ID NO: 624); WSNCHWNYVHIFLDC (SEQ ID NO: 625);DRSCTWNYVRISYDC (SEQ ID NO: 626); SGSCKWDYVHIFLDC (SEQ ID NO: 627);SRSCIWNYAHIHLDC (SEQ ID NO: 628); SMSCYWQYERIFLDC (SEQ ID NO: 629);YRSCNWNYVSIFLDC (SEQ ID NO: 630); SGSCKWDYVHIFLDC (SEQ ID NO: 631);YKSCHWDYVHIFLDC (SEQ ID NO: 632); YGSCTWNYVHIFMEC (SEQ ID NO: 633);FSSCNWNYVHIFLDC (SEQ ID NO: 634); WRSCNWNYAHIFLDC (SEQ ID NO: 635);YGSCQWNYVHIFLDC (SEQ ID NO: 636); YRSCNWNYVHIFLDC (SEQ ID NO: 637);NMSCHWDYVHIFLDC (SEQ ID NO: 638); FGPCTWNYARISWDC (SEQ ID NO: 639);XXsCXWXYvhIfXdC (SEQ ID NO: 640); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 641);RDTGGQCRWDYVHIFMDC (SEQ ID NO: 642); AGVPAGCTWNYVHIFMEC (SEQ ID NO:643); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 644); DGGPAGCSWNYVHIFMEC (SEQ IDNO: 645); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 646); CTWNYVHIFMDCGEGEGP (SEQID NO: 647); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 648); AEVPAGCWWNYVHIFMEC(SEQ ID NO: 649); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 650);SGASGGCKWNYVHIFMDC (SEQ ID NO: 651); TPGCRWNYVHIFMECEAL (SEQ ID NO:652); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 653); PGAFDIPFPAHWVPNT (SEQ ID NO:654); RGACDIPFPAHWIPNT (SEQ ID NO: 655); QGDFDIPFPAHWVPIT (SEQ ID NO:656); XGafDIPFPAHWvPnT (SEQ ID NO: 657); RGDGNDSDIPFPAHWVPRT (SEQ ID NO:658); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 659); WAGGNDCDIPFPAHWIPNT (SEQ IDNO: 660); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 661); AGSGNDSDIPFPAHWVPRT (SEQID NO: 662); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 663); and/orRECGRCGDIPFPAHWVPRT (SEQ ID NO: 664).

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the target is at least 5,10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orbetween 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times greater than the K_(d) ofthe AB not modified with an MM or of the parental AB towards the target.Conversely, the binding affinity of the AB modified with a MM towardsthe target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000,10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000,10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB notmodified with an MM or of the parental AB towards the target.

The dissociation constant (K_(d)) of the MM towards the AB is generallygreater than the K_(d) of the AB towards the target. The K_(d) of the MMtowards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 timesgreater than the K_(d) of the AB towards the target. Conversely, thebinding affinity of the MM towards the AB is generally lower than thebinding affinity of the AB towards the target. The binding affinity ofMM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lowerthan the binding affinity of the AB towards the target.

When the AB is modified with a MM and is in the presence of the targetspecific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target. When compared tothe binding of the AB not modified with an MM or the binding of theparental AB to the target the AB's ability to bind the target whenmodified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months or more when measured in vivo or in an in vitro assay.

The MM inhibits the binding of the AB to the target. The MM binds theantigen binding domain of the AB and inhibits binding of the AB to thetarget. The MM can sterically inhibit the binding of the AB to thetarget. The MM can allosterically inhibit the binding of the AB to itstarget. In these embodiments when the AB is modified or coupled to a MMand in the presence of target there is no binding or substantially nobinding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 50% binding of the AB to the target, as compared to the binding ofthe AB not modified with an MM, the parental AB, or the AB not coupledto an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48,60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer whenmeasured in vivo or in an in vitro assay.

When an AB is coupled to or modified by a MM, the MM ‘masks’ or reducesor otherwise inhibits the specific binding of the AB to the target. Whenan AB is coupled to or modified by a MM, such coupling or modificationcan effect a structural change that reduces or inhibits the ability ofthe AB to specifically bind its target.

An AB coupled to or modified with an MM can be represented by thefollowing formulae (in order from an amino (N) terminal region tocarboxyl (C) terminal region:

(MM)-(AB)

(AB)-(MM)

(MM)-L-(AB)

(AB)-L-(MM)

where MM is a masking moiety, the AB is an antibody or antibody fragmentthereof, and the L is a linker. In many embodiments, it may be desirableto insert one or more linkers, e.g., flexible linkers, into thecomposition so as to provide for flexibility.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments, the MM contains no or substantially no homologyto any natural binding partner of the AB. In some embodiments, the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 25% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 50% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In some embodiments, the activatable antibodies include an AB that ismodified by an MM and also includes one or more cleavable moieties (CM).Such activatable antibodies exhibit activatable/switchable binding, tothe AB's target. Activatable antibodies generally include an antibody orantibody fragment (AB), modified by or coupled to a masking moiety (MM)and a modifiable or cleavable moiety (CM). In some embodiments, the CMcontains an amino acid sequence that serves as a substrate for at leastone protease selected from matriptase and uPA.

The elements of the activatable antibodies are arranged so that the MMand CM are positioned such that in a cleaved (or relatively active)state and in the presence of a target, the AB binds a target while in anuncleaved (or relatively inactive) state in the presence of the target,specific binding of the AB to its target is reduced or inhibited. Thespecific binding of the AB to its target can be reduced due to theinhibition or masking of the AB's ability to specifically bind itstarget by the MM.

The K_(d) of the AB modified with a MM and a CM towards the target is atleast 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000,100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 orgreater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 timesgreater than the K_(d) of the AB not modified with an MM and a CM or ofthe parental AB towards the target. Conversely, the binding affinity ofthe AB modified with a MM and a CM towards the target is at least 5, 10,25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orbetween 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times lower than the bindingaffinity of the AB not modified with an MM and a CM or of the parentalAB towards the target.

When the AB is modified with a MM and a CM and is in the presence of thetarget but not in the presence of a modifying agent (for example atleast one protease selected from matriptase and uPA), specific bindingof the AB to its target is reduced or inhibited, as compared to thespecific binding of the AB not modified with an MM and a CM or of theparental AB to the target. When compared to the binding of the parentalAB or the binding of an AB not modified with an MM and a CM to itstarget, the AB's ability to bind the target when modified with an MM anda CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28,24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90,120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months or longer when measured in vivo or in an in vitro assay.

As used herein, the term cleaved state refers to the condition of theactivatable antibodies following modification of the CM by at least oneprotease selected from matriptase and uPA. The term uncleaved state, asused herein, refers to the condition of the activatable antibodies inthe absence of cleavage of the CM by a protease selected from matriptaseand uPA. As discussed above, the term “activatable antibodies” is usedherein to refer to an activatable antibody in both its uncleaved(native) state, as well as in its cleaved state. It will be apparent tothe ordinarily skilled artisan that in some embodiments a cleavedactivatable antibody may lack an MM due to cleavage of the CM byprotease, resulting in release of at least the MM (e.g., where the MM isnot joined to the activatable antibodies by a covalent bond (e.g., adisulfide bond between cysteine residues).

By activatable or switchable is meant that the activatable antibodyexhibits a first level of binding to a target when in a inhibited,masked or uncleaved state (i.e., a first conformation), and a secondlevel of binding to the target in the uninhibited, unmasked and/orcleaved state (i.e., a second conformation), where the second level oftarget binding is greater than the first level of binding. In general,the access of target to the AB of the activatable antibody is greater inthe presence of a cleaving agent capable of cleaving the CM, i.e., aprotease selected from matriptase and uPA, than in the absence of such acleaving agent. Thus, when the activatable antibody is in the uncleavedstate, the AB is inhibited from target binding and can be masked fromtarget binding (i.e., the first conformation is such the AB cannot bindthe target), and in the cleaved state the AB is not inhibited or isunmasked to target binding.

The CM and AB of the activatable antibodies are selected so that the ABrepresents a binding moiety for a given target, and the CM represents asubstrate for a protease selected from matriptase and uPA. In someembodiments, the protease is co-localized with the target at a treatmentsite or diagnostic site in a subject. As used herein, co-localizedrefers to being at the same site or relatively close nearby. In someembodiments, a protease cleaves a CM yielding an activated antibody thatbinds to a target located nearby the cleavage site. The activatableantibodies disclosed herein find particular use where, for example, aprotease capable of cleaving a site in the CM, i.e., a protease selectedfrom matriptase and uPA, is present at relatively higher levels intarget-containing tissue of a treatment site or diagnostic site than intissue of non-treatment sites (for example in healthy tissue). In someembodiments, a CM of the disclosure is also cleaved by one or more otherproteases. In some embodiments, it is the one or more other proteasesthat is co-localized with the target and that is responsible forcleavage of the CM in vivo.

In some embodiments activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe AB at non-treatment sites if the AB were not masked or otherwiseinhibited from binding to the target.

In general, an activatable antibody can be designed by selecting an ABof interest and constructing the remainder of the activatable antibodyso that, when conformationally constrained, the MM provides for maskingof the AB or reduction of binding of the AB to its target. Structuraldesign criteria can be to be taken into account to provide for thisfunctional feature.

Activatable antibodies exhibiting a switchable phenotype of a desireddynamic range for target binding in an inhibited versus an uninhibitedconformation are provided. Dynamic range generally refers to a ratio of(a) a maximum detected level of a parameter under a first set ofconditions to (b) a minimum detected value of that parameter under asecond set of conditions. For example, in the context of an activatableantibody, the dynamic range refers to the ratio of (a) a maximumdetected level of target protein binding to an activatable antibody inthe presence of at least one protease selected from matriptase and uPAcapable of cleaving the CM of the activatable antibodies to (b) aminimum detected level of target protein binding to an activatableantibody in the absence of the protease. The dynamic range of anactivatable antibody can be calculated as the ratio of the equilibriumdissociation constant of an activatable antibody cleaving agent (e.g.,enzyme) treatment to the equilibrium dissociation constant of theactivatable antibodies cleaving agent treatment. The greater the dynamicrange of an activatable antibody, the better the switchable phenotype ofthe activatable antibody. Activatable antibodies having relativelyhigher dynamic range values (e.g., greater than 1) exhibit moredesirable switching phenotypes such that target protein binding by theactivatable antibodies occurs to a greater extent (e.g., predominantlyoccurs) in the presence of a cleaving agent (e.g., enzyme) capable ofcleaving the CM of the activatable antibodies than in the absence of acleaving agent.

Activatable antibodies can be provided in a variety of structuralconfigurations. Exemplary formulae for activatable antibodies areprovided below. It is specifically contemplated that the N- toC-terminal order of the AB, MM and CM may be reversed within anactivatable antibody. It is also specifically contemplated that the CMand MM may overlap in amino acid sequence, e.g., such that the CM iscontained within the MM.

For example, activatable antibodies can be represented by the followingformula (in order from an amino (N) terminal region to carboxyl (C)terminal region:

(MM)-(CM)-(AB)

(AB)-(CM)-(MM)

where MM is a masking moiety, CM is a cleavable moiety, and AB is anantibody or fragment thereof. It should be noted that although MM and CMare indicated as distinct components in the formulae above, in allexemplary embodiments (including formulae) disclosed herein it iscontemplated that the amino acid sequences of the MM and the CM couldoverlap, e.g., such that the CM is completely or partially containedwithin the MM. In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments, the MM contains no or substantially no homologyto any natural binding partner of the AB. In some embodiments, the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 50% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 25% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In many embodiments it may be desirable to insert one or more linkers,e.g., flexible linkers, into the activatable antibody construct so as toprovide for flexibility at one or more of the MM-CM junction, the CM-ABjunction, or both. For example, the AB, MM, and/or CM may not contain asufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Glyand Ser, particularly Gly) to provide the desired flexibility. As such,the switchable phenotype of such activatable antibody constructs maybenefit from introduction of one or more amino acids to provide for aflexible linker. In addition, as described below, where the activatableantibody is provided as a conformationally constrained construct, aflexible linker can be operably inserted to facilitate formation andmaintenance of a cyclic structure in the uncleaved activatable antibody.

For example, in certain embodiments an activatable antibody comprisesone of the following formulae (where the formula below represent anamino acid sequence in either N- to C-terminal direction or C- toN-terminal direction):

(MM)-L1-(CM)-(AB)

(MM)-(CM)-L2-(AB)

(MM)-L1-(CM)-L2-(AB)

wherein MM, CM, and AB are as defined above; wherein L1 and L2 are eachindependently and optionally present or absent, are the same ordifferent flexible linkers that include at least 1 flexible amino acid(e.g., Gly). In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements. Examples include, but are notlimited to, targeting moieties (e.g., a ligand for a receptor of a cellpresent in a target tissue) and serum half-life extending moieties(e.g., polypeptides that bind serum proteins, such as immunoglobulin(e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)).

The CM is specifically cleaved by at least one protease selected frommatriptase and uPA at a rate of about 0.001-1500×10⁴ M⁻¹S⁻¹ or at least0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50,75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10⁴ M⁻¹S⁻¹.In some embodiments, the CM is specifically cleaved at a rate of about100,000 M⁻¹S⁻¹. In some embodiments, the CM is specifically cleaved at arate from about 1×10E2 to about 1×10E6 M⁻¹S⁻¹ (i.e., from about 1×10² toabout 1×10⁶ M⁻¹S⁻¹).

For specific cleavage by an enzyme, contact between the enzyme and CM ismade. When the activatable antibody comprising an AB coupled to a MM anda CM is in the presence of target and sufficient enzyme activity, the CMcan be cleaved. Sufficient enzyme activity can refer to the ability ofthe enzyme to make contact with the CM and effect cleavage. It canreadily be envisioned that an enzyme may be in the vicinity of the CMbut unable to cleave because of other cellular factors or proteinmodification of the enzyme.

Linkers suitable for use in compositions described herein are generallyones that provide flexibility of the modified AB or the activatableantibodies to facilitate the inhibition of the binding of the AB to thetarget. Such linkers are generally referred to as flexible linkers.Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length.

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 385) and(GGGS)n (SEQ ID NO: 386), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers arerelatively unstructured, and therefore may be able to serve as a neutraltether between components. Glycine accesses significantly more phi-psispace than even alanine, and is much less restricted than residues withlonger side chains (see Scheraga, Rev. Computational Chem. 11173-142(1992)). Exemplary flexible linkers include, but are not limited toGly-Gly-Ser-Gly (SEQ ID NO: 387), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 388),Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 389), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:390), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 391), Gly-Ser-Ser-Ser-Gly (SEQ IDNO: 392), and the like. The ordinarily skilled artisan will recognizethat design of an activatable antibodies can include linkers that areall or partially flexible, such that the linker can include a flexiblelinker as well as one or more portions that confer less flexiblestructure to provide for a desired activatable antibodies structure.

In some embodiments, the activatable antibodies described herein alsoinclude an agent conjugated to the activatable antibody. In someembodiments, the conjugated agent is a therapeutic agent, such as ananti-inflammatory and/or an antineoplastic agent. In such embodiments,the agent is conjugated to a carbohydrate moiety of the activatableantibody, for example, in some embodiments, where the carbohydratemoiety is located outside the antigen-binding region of the antibody orantigen-binding fragment in the activatable antibody. In someembodiments, the agent is conjugated to a sulfhydryl group of theantibody or antigen-binding fragment in the activatable antibody.

In some embodiments, the agent is a cytotoxic agent such as a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

In some embodiments, the agent is a detectable moiety such as, forexample, a label or other marker. For example, the agent is or includesa radiolabeled amino acid, one or more biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), one or more radioisotopes or radionuclides, oneor more fluorescent labels, one or more enzymatic labels, and/or one ormore chemiluminescent agents. In some embodiments, detectable moietiesare attached by spacer molecules.

The disclosure also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablecytotoxic agents include, for example, dolastatins and derivativesthereof (e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). Forexample, the agent is monomethyl auristatin E (MMAE) or monomethylauristatin D (MMAD). In some embodiments, the agent is an agent selectedfrom the group listed in Table 3. In some embodiments, the agent is adolastatin. In some embodiments, the agent is an auristatin orderivative thereof. In some embodiments, the agent is auristatin E or aderivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some embodiments, the agent is linked to the AB using a maleimidecaproyl-valine-citrulline linker or a maleimide PEG-valine-citrullinelinker. In some embodiments, the agent is linked to the AB using amaleimide caproyl-valine-citrulline linker. In some embodiments, theagent is linked to the AB using a maleimide PEG-valine-citrulline linkerIn some embodiments, the agent is monomethyl auristatin D (MMAD) linkedto the AB using a maleimidePEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and thislinker payload construct is referred to herein as “vc-MMAD.” In someembodiments, the agent is monomethyl auristatin E (MMAE) linked to theAB using a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker, and this linker payload construct is referred to herein as“vc-MMAE.” The structures of vc-MMAD and vc-MMAE are shown below:

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Table 3 lists some of the exemplary pharmaceutical agents that may beemployed in the herein described disclosure but in no way is meant to bean exhaustive list.

TABLE 3 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTSTurbostatin Auristatins Phenstatins Auristatin E HydroxyphenstatinMonomethyl auristatin D (MMAD) Spongistatin 5 Monomethyl auristatin E(MMAE) Spongistatin 7 Desmethyl auristatin E (DMAE) Halistatin 1Auristatin F Halistatin 2 Monomethyl auristatin F (MMAF) Halistatin 3Desmethyl auristatin F (DMAF) Modified Bryostatins Auristatinderivatives, e.g., amides thereof Halocomstatins Auristatin tyraminePyrrolobenzimidazoles (PBI) Auristatin quinoline Cibrostatin6Dolastatins Doxaliform Dolastatin derivatives Anthracyclins analoguesDolastatin 16 DmJ Cemadotin analogue (CemCH2-SH) Dolastatin 16 DpvPseudomonas toxin A (PE38) variant Maytansinoids, e.g. DM-1; DM-4Pseudomonas toxin A (ZZ-PE38) variant Maytansinoid derivatives ZJ-101Duocarmycin OSW-1 Duocarmycin derivatives 4-NitrobenzyloxycarbonylDerivatives of Alpha-amanitin O6-Benzylguanine AnthracyclinesTopoisomerase inhibitors Doxorubicin Hemiasterlin DaunorubicinCephalotaxine Bryostatins Homoharringtonine CamptothecinPyrrolobenzodiazepine dimers (PBDs) Camptothecin derivativesFunctionalized pyrrolobenzodiazepenes 7-substituted CamptothecinCalicheamicins 10,11- PodophyllotoxinsDifluoromethylenedioxycamptothecin Taxanes Combretastatins Vincaalkaloids Debromoaplysiatoxin CONJUGATABLE DETECTION Kahalalide-FREAGENTS Discodermolide Fluorescein and derivatives thereofEcteinascidins Fluorescein isothiocyanate (FITC) ANTIVIRALSRADIOPHARMACEUTICALS Acyclovir ¹²⁵I Vira A ¹³¹I Symmetrel ⁸⁹ZrANTIFUNGALS ¹¹¹In Nystatin ¹²³I ADDITIONAL ANTI-NEOPLASTICS ¹³¹IAdriamycin ⁹⁹mTc Cerubidine ²⁰¹Tl Bleomycin ¹³³Xe Alkeran ¹¹C Velban⁶²Cu Oncovin ¹⁸F Fluorouracil ⁶⁸Ga Methotrexate ¹³N Thiotepa ¹⁵OBisantrene ³⁸K Novantrone ⁸²Rb Thioguanine ⁹⁹mTc (Technetium)Procarabizine HEAVY METALS Cytarabine Barium ANTI-BACTERIALS GoldAminoglycosides Platinum Streptomycin ANTI-MYCOPLASMALS NeomycinTylosine Kanamycin Spectinomycin Amikacin Gentamicin TobramycinStreptomycin B Spectinomycin Ampicillin Sulfanilamide PolymyxinChloramphenicol

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of thedisclosure. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. In some embodiments,the binding is, however, covalent binding. Covalent binding can beachieved either by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present disclosure, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

In some embodiments, in addition to the compositions and methodsprovided herein, the conjugated activatable antibody can also bemodified for site-specific conjugation through modified amino acidsequences inserted or otherwise included in the activatable antibodysequence. These modified amino acid sequences are designed to allow forcontrolled placement and/or dosage of the conjugated agent within aconjugated activatable antibody. For example, the activatable antibodycan be engineered to include cysteine substitutions at positions onlight and heavy chains that provide reactive thiol groups and do notnegatively impact protein folding and assembly, nor alter antigenbinding. In some embodiments, the activatable antibody can be engineeredto include or otherwise introduce one or more non-natural amino acidresidues within the activatable antibody to provide suitable sites forconjugation. In some embodiments, the activatable antibody can beengineered to include or otherwise introduce enzymatically activatablepeptide sequences within the activatable antibody sequence.

Suitable linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. In someembodiments, suitable linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii)SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC. Additional linkers include, but arenot limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NETS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, i.e., cleavable ornon-cleavable, or the two or more linkers are different, i.e., at leastone cleavable and at least one non-cleavable.

The present disclosure utilizes several methods for attaching agents toABs: (a) attachment to the carbohydrate moieties of the AB, or (b)attachment to sulfhydryl groups of the AB, or (c) attachment to aminogroups of the AB, or (d) attachment to carboxylate groups of the AB.According to the disclosure, ABs may be covalently attached to an agentthrough an intermediate linker having at least two reactive groups, oneto react with AB and one to react with the agent. The linker, which mayinclude any compatible organic compound, can be chosen such that thereaction with AB (or agent) does not adversely affect AB reactivity andselectivity. Furthermore, the attachment of linker to agent might notdestroy the activity of the agent. Suitable linkers for reaction withoxidized antibodies or oxidized antibody fragments include thosecontaining an amine selected from the group consisting of primary amine,secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present disclosure, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present disclosure, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the primary amino groups present inunmodified lysine residues in the Ab. Such reactive groups include, butare not limited to, NHS carboxylic or carbonic esters, sulfo-NHScarboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonicesters, pentafluorophenyl carboxylic or carbonic esters, acylimidazoles, isocyanates, and isothiocyanates.

According to the present disclosure, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the carboxylic acid groups present inaspartate or glutamate residues in the Ab, which have been activatedwith suitable reagents. Suitable activating reagents include EDC, withor without added NHS or sulfo-NHS, and other dehydrating agents utilizedfor carboxamide formation. In these instances, the functional groupspresent in the suitable linkers would include primary and secondaryamines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker. In someembodiments, the AB is first attached to the MM, CM and associatedlinkers and then attached to the linker for conjugation purposes.

Branched Linkers:

In specific embodiments, branched linkers that have multiple sites forattachment of agents are utilized. For multiple site linkers, a singlecovalent attachment to an AB would result in an AB-linker intermediatecapable of binding an agent at a number of sites. The sites may bealdehyde or sulfhydryl groups or any chemical site to which agents canbe attached.

In some embodiments, higher specific activity (or higher ratio of agentsto AB) can be achieved by attachment of a single site linker at aplurality of sites on the AB. This plurality of sites may be introducedinto the AB by either of two methods. First, one may generate multiplealdehyde groups and/or sulfhydryl groups in the same AB. Second, one mayattach to an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

Cleavable Linkers:

Peptide linkers that are susceptible to cleavage by enzymes of thecomplement system, such as but not limited to u-plasminogen activator,tissue plasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentdisclosure. According to one method of the present disclosure, an agentis attached via a linker susceptible to cleavage by complement. Theantibody is selected from a class that can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present disclosure, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as au-plasminogen activator, a tissue plasminogen activator, plasmin, ortrypsin. These cleavable linkers are useful in conjugated activatableantibodies that include an extracellular toxin, e.g., by way ofnon-limiting example, any of the extracellular toxins shown in Table 3.

Non-limiting examples of cleavable linker sequences are provided inTable 4.

TABLE 4 Exemplary Linker Sequences for Conjugation Types of CleavableSequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinasePRFKIIGG (SEQ ID NO: 665) PRFRIIGG (SEQ ID NO: 666) TGFβ SSRHRRALD (SEQID NO: 667) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 668) StaphylokinaseSSSFDKGKYKKGDDA (SEQ ID NO: 669) SSSFDKGKYKRGDDA (SEQ ID NO: 670) FactorXa cleavable sequences IEGR (SEQ ID NO: 671) IDGR (SEQ ID NO: 672)GGSIDGR (SEQ ID NO: 673) MMP cleavable sequences Gelatinase A PLGLWA(SEQ ID NO: 674) Collagenase cleavable sequences Calf skin collagen(α1(I) chain) GPQGIAGQ (SEQ ID NO: 675) Calf skin collagen (α2(I) chain)GPQGLLGA (SEQ ID NO: 676) Bovine cartilage collagen (α1(II) chain) GIAGQ(SEQ ID NO: 677) Human liver collagen (α1(III) chain) GPLGIAGI (SEQ IDNO: 678) Human α₂M GPEGLRVG (SEQ ID NO: 679) Human PZP YGAGLGVV (SEQ IDNO: 680) AGLGVVER (SEQ ID NO: 681) AGLGISST (SEQ ID NO: 682) Rat α₁MEPQALAMS (SEQ ID NO: 683) QALAMSAI (SEQ ID NO: 312) Rat α₂M AAYHLVSQ(SEQ ID NO: 315) MDAFLESS (SEQ ID NO: 316) Rat α₁I₃(2J) ESLPVVAV (SEQ IDNO: 317) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 318) Human fibroblastcollagenase DVAQFVLT (SEQ ID NO: 319) (autolytic cleavages) VAQFVLTE(SEQ ID NO: 372) AQFVLTEG (SEQ ID NO: 373) PVQPIGPQ (SEQ ID NO: 380)

In addition, agents may be attached via disulfide bonds (for example,the disulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In some embodiments, the reducing agent that wouldmodify a CM would also modify the linker of the conjugated activatableantibody.

Spacers and Cleavable Elements:

In some embodiments, it may be necessary to construct the linker in sucha way as to optimize the spacing between the agent and the AB of theactivatable antibody. This may be accomplished by use of a linker of thegeneral structure:

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In some embodiments, the linker may comprise a spacer element and acleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be particular mode of attachment orreaction. Any reaction providing a product of suitable stability andbiological compatibility is acceptable.

Serum Complement and Selection of Linkers:

According to one method of the present disclosure, when release of anagent is desired, an AB that is an antibody of a class that can activatecomplement is used. The resulting conjugate retains both the ability tobind antigen and activate the complement cascade. Thus, according tothis embodiment of the present disclosure, an agent is joined to one endof the cleavable linker or cleavable element and the other end of thelinker group is attached to a specific site on the AB. For example, ifthe agent has an hydroxy group or an amino group, it may be attached tothe carboxy terminus of a peptide, amino acid or other suitably chosenlinker via an ester or amide bond, respectively. For example, suchagents may be attached to the linker peptide via a carbodimide reaction.If the agent contains functional groups that would interfere withattachment to the linker, these interfering functional groups can beblocked before attachment and deblocked once the product conjugate orintermediate is made. The opposite or amino terminus of the linker isthen used either directly or after further modification for binding toan AB that is capable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the activatable antibody. The other end of the linker or spacerelement may be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond that attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 3.

Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agentwithout complement activation is desired since activation of thecomplement cascade will ultimately lyse the target cell. Hence, thisapproach is useful when delivery and release of the agent should beaccomplished without killing the target cell. Such is the goal whendelivery of cell mediators such as hormones, enzymes, corticosteroids,neurotransmitters, genes or enzymes to target cells is desired. Theseconjugates may be prepared by attaching the agent to an AB that is notcapable of activating complement via a linker that is mildly susceptibleto cleavage by serum proteases. When this conjugate is administered toan individual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

Biochemical Cross Linkers:

In some embodiments, the activatable antibody may be conjugated to oneor more therapeutic agents using certain biochemical cross-linkers.Cross-linking reagents form molecular bridges that tie togetherfunctional groups of two different molecules. To link two differentproteins in a step-wise manner, hetero-bifunctional cross-linkers can beused that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, forexample, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labilelinkers cleavable in the low-pH environment of the lysosome may be used,for example: bis-sialyl ether. Other suitable linkers includecathepsin-labile substrates, particularly those that show optimalfunction at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 5.

TABLE 5 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length after cross-linking Linker ReactiveToward Advantages and Applications (Angstroms) SMPT Primary aminesGreater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation 6.8Å Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extendedspacer arm 15.6 Å Sulfhydryls Sulfo-LC-SPDP Primary amines Extenderspacer arm 15.6 Å Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugationHapten-carrier protein conjugation Sulfo-SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibodyconjugation 9.9 Å Sulfhydryls Hapten-carrier protein conjugationSulfo-MBS Primary amines Water-soluble 9.9 Å Sulfhydryls SIAB Primaryamines Enzyme-antibody conjugation 10.6 Å Sulfhydryls Sulfo-SIAB Primaryamines Water-soluble 10.6 Å Sulfhydryls SMPB Primary amines Extendedspacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPBPrimary amines Extended spacer arm 14.5 Å Sulfhydryls Water-solubleEDE/Sulfo-NHS Primary amines Hapten-Carrier conjugation 0 Carboxylgroups ABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective

Non-Cleavable Linkers or Direct Attachment:

In some embodiments of the disclosure, the conjugate may be designed sothat the agent is delivered to the target but not released. This may beaccomplished by attaching an agent to an AB either directly or via anon-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds that may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A-general formula for such an organiclinker could be

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

Non-Cleavable Conjugates:

In some embodiments, a compound may be attached to ABs that do notactivate complement. When using ABs that are incapable of complementactivation, this attachment may be accomplished using linkers that aresusceptible to cleavage by activated complement or using linkers thatare not susceptible to cleavage by activated complement.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present disclosure canbe conjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The term “a”entity or “an” entity refers to one or more of that entity. For example,a compound refers to one or more compounds. As such, the terms “a”,“an”, “one or more” and “at least one” can be used interchangeably.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; in some embodiments, ≦100 nM and in some embodiments, ≦10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present disclosure is said to specifically bind to thetarget, when the equilibrium binding constant (K_(d)) is ≦1 μM, in someembodiments ≦100 nM, in some embodiments ≦10 nM, and in some embodiments≦100 pM to about 1 pM, as measured by assays such as radioligand bindingassays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the disclosure include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the disclosure comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. In some embodiments, oligonucleotides are10 to 60 bases in length and in some embodiments, 12, 13, 14, 15, 16,17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usuallysingle stranded, e.g., for probes, although oligonucleotides may bedouble stranded, e.g., for use in the construction of a gene mutant.Oligonucleotides of the disclosure are either sense or antisenseoligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Green, Eds., Sinauer Associates,Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present disclosure. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, in some embodiments, at least 90 percent sequenceidentity, in some embodiments, at least 95 percent sequence identity,and in some embodiments, at least 99 percent sequence identity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, in some embodiments, atleast 80%, 90%, 95%, and in some embodiments, 99%. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic amino acids areaspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Suitable amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. In some embodiments,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the disclosure.

Suitable amino acid substitutions are those that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions (forexample, conservative amino acid substitutions) may be made in thenaturally-occurring sequence (for example, in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, in some embodiments, at least 14 amino acids long, in someembodiments, at least 20 amino acids long, usually at least 50 aminoacids long, and in some embodiments, at least 70 amino acids long. Theterm “analog” as used herein refers to polypeptides that are comprisedof a segment of at least 25 amino acids that has substantial identity toa portion of a deduced amino acid sequence and that has specific bindingto the target, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, in some embodiments, atleast 50 amino acids long or longer, and can often be as long as afull-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and in someembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, in some embodiments, more than about 85%, 90%, 95%, and99%. In some embodiments, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Activatable antibodies of the disclosure specifically bind a giventarget, e.g., a human target protein. Also included in the disclosureare activatable antibodies that bind to the same epitope as theactivatable antibodies described herein.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., a murine monoclonal or humanized antibody) has the samespecificity as a monoclonal antibody used in the methods describedherein by ascertaining whether the former prevents the latter frombinding to the target. If the monoclonal antibody being tested competeswith the monoclonal antibody of the disclosure, as shown by a decreasein binding by the monoclonal antibody of the disclosure, then the twomonoclonal antibodies bind to the same, or a closely related, epitope.An alternative method for determining whether a monoclonal antibody hasthe specificity of a monoclonal antibody of the disclosure is topre-incubate the monoclonal antibody of the disclosure with the targetand then add the monoclonal antibody being tested to determine if themonoclonal antibody being tested is inhibited in its ability to bind thetarget. If the monoclonal antibody being tested is inhibited then, inall likelihood, it has the same, or functionally equivalent, epitopicspecificity as the monoclonal antibody of the disclosure.

Multispecific Activatable Antibodies

The disclosure also provides multispecific activatable antibodies. Themultispecific activatable antibodies provided herein are multispecificantibodies that recognize two or more different antigens or epitopes andthat include at least one masking moiety (MM) linked to at least oneantigen- or epitope-binding domain of the multispecific antibody suchthat coupling of the MM reduces the ability of the antigen- orepitope-binding domain to bind its target. In some embodiments, the MMis coupled to the antigen- or epitope-binding domain of themultispecific antibody via a cleavable moiety (CM) that functions as asubstrate for at least one protease selected from uPA and matriptase.The activatable multispecific antibodies provided herein are stable incirculation, activated at intended sites of therapy and/or diagnosis butnot in normal, i.e., healthy tissue, and, when activated, exhibitbinding to a target that is at least comparable to the corresponding,unmodified multispecific antibody.

In some embodiments, the multispecific activatable antibodies aredesigned to engage immune effector cells, also referred to herein asimmune-effector cell engaging multispecific activatable antibodies. Insome embodiments, the multispecific activatable antibodies are designedto engage leukocytes, also referred to herein as leukocyte engagingmultispecific activatable antibodies. In some embodiments, themultispecific activatable antibodies are designed to engage T cells,also referred to herein as T-cell engaging multispecific activatableantibodies. In some embodiments, the multispecific activatableantibodies engage a surface antigen on a leukocyte, such as on a T cell,on a natural killer (NK) cell, on a myeloid mononuclear cell, on amacrophage, and/or on another immune effector cell. In some embodiments,the immune effector cell is a leukocyte. In some embodiments, the immuneeffector cell is a T cell. In some embodiments, the immune effector cellis a NK cell. In some embodiments, the immune effector cell is amononuclear cell, such as a myeloid mononuclear cell. In someembodiments, the multispecific activatable antibodies are designed tobind or otherwise interact with more than one target and/or more thanone epitope, also referred to herein as multi-antigen targetingactivatable antibodies. As used herein, the terms “target” and “antigen”are used interchangeably.

In some embodiments, immune effector cell engaging multispecificactivatable antibodies of the disclosure include a targeting antibody orantigen-binding fragment thereof and an immune effector cell engagingantibody or antigen-binding portion thereof, where at least one of thetargeting antibody or antigen-binding fragment thereof and/or the immuneeffector cell engaging antibody or antigen-binding portion thereof ismasked. In some embodiments, the immune effector cell engaging antibodyor antigen binding fragment thereof includes a first antibody orantigen-binding fragment thereof (AB1) that binds a first, immuneeffector cell engaging target, where the AB1 is attached to a maskingmoiety (MM1) such that coupling of the MM1 reduces the ability of theAB1 to bind the first target. In some embodiments, the targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second target, where the AB2 isattached to a masking moiety (MM2) such that coupling of the MM2 reducesthe ability of the AB2 to bind the second target. In some embodiments,the immune effector cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, immune effector cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target, and thetargeting antibody or antigen-binding fragment thereof includes a secondantibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target. In someembodiments, the non-immune effector cell engaging antibody is a cancertargeting antibody. In some embodiments the non-immune cell effectorantibody is an IgG. In some embodiments the immune effector cellengaging antibody is a scFv. In some embodiments the targeting antibody(e.g., non-immune cell effector antibody) is an IgG and the immuneeffector cell engaging antibody is a scFv. In some embodiments, theimmune effector cell is a leukocyte. In some embodiments, the immuneeffector cell is a T cell. In some embodiments, the immune effector cellis a NK cell. In some embodiments, the immune effector cell is a myeloidmononuclear cell.

In some embodiments, T-cell engaging multispecific activatableantibodies of the disclosure include a targeting antibody orantigen-binding fragment thereof and a T-cell engaging antibody orantigen-binding portion thereof, where at least one of the targetingantibody or antigen-binding fragment thereof and/or the T-cell engagingantibody or antigen-binding portion thereof is masked. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target. In some embodiments,the targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target, and the targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second target, where the AB2 isattached to a masking moiety (MM2) such that coupling of the MM2 reducesthe ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibodies include a cancer targeting antibody or antigen-bindingfragment thereof and a T-cell engaging antibody or antigen-bindingportion thereof, where at least one of the cancer targeting antibody orantigen-binding fragment thereof and/or the T-cell engaging antibody orantigen-binding portion thereof is masked. In some embodiments, theT-cell engaging antibody or antigen binding fragment thereof includes afirst antibody or antigen-binding fragment thereof (AB1) that binds afirst, T-cell engaging target, where the AB1 is attached to a maskingmoiety (MM1) such that coupling of the MM1 reduces the ability of theAB1 to bind the first target. In some embodiments, the cancer targetingantibody or antigen-binding fragment thereof includes a second antibodyor fragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second, cancer-related target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second, cancer-relatedtarget. In some embodiments, the T-cell engaging antibody or antigenbinding fragment thereof includes a first antibody or antigen-bindingfragment thereof (AB1) that binds a first, T-cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target, and thecancer targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibodies include a cancer targeting IgG antibody or antigen-bindingfragment thereof and a T-cell engaging scFv, where at least one of thecancer targeting IgG antibody or antigen-binding fragment thereof and/orthe T-cell engaging antibody or antigen-binding portion thereof ismasked. In some embodiments, the T-cell engaging antibody or antigenbinding fragment thereof includes a first antibody or antigen-bindingfragment thereof (AB1) that binds a first, T-cell engaging target, wherethe AB1 is attached to a masking moiety (MM1) such that coupling of theMM1 reduces the ability of the AB1 to bind the first target. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the T-cell engaging antibody or antigen binding fragmentthereof includes a first antibody or antigen-binding fragment thereof(AB1) that binds a first, T-cell engaging target, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind the first target, and the cancertargeting IgG antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments of an immune effector cell engaging multi specificactivatable antibody, one antigen is typically an antigen present on thesurface of a tumor cell or other cell type associated with disease, suchas, but not limited to, any target listed in Table 1, such as, but notlimited to, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71(transferrin receptor), and another antigen is typically a stimulatoryor inhibitory receptor present on the surface of a T-cell, naturalkiller (NK) cell, myeloid mononuclear cell, macrophage, and/or otherimmune effector cell, such as, but not limited to, B7-H4, BTLA, CD3,CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR,HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In someembodiments, the antigen is a stimulatory receptor present on thesurface of a T cell or NK cell; examples of such stimulatory receptorsinclude, but are not limited to, CD3, CD27, CD28, CD137 (also referredto as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments,the antigen is an inhibitory receptor present on the surface of aT-cell; examples of such inhibitory receptors include, but are notlimited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressedKIRs. The antibody domain conferring specificity to the T-cell surfaceantigen may also be substituted by a ligand or ligand domain that bindsto a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/orother immune effector cell receptor, such as, but not limited to, B7-1,B7-2, B7H3, PD-L1, PD-L2, or TNFSF9.

One embodiment of the disclosure is a multispecific activatable antibodythat is activatable in a cancer microenvironment and that includes anantibody, for example a IgG or scFv, directed to a tumor target and anagonist antibody, for example an IgG or scFv, directed to aco-stimulatory receptor expressed on the surface of an activated T cellor NK cell, wherein at least one of the cancer target antibody and/oragonist antibody is masked. Examples of co-stimulatory receptorsinclude, but are not limited to, CD27, CD137, GITR, HVEM, NKG2D, andOX40. In this embodiment, the multispecific activatable antibody, onceactivated by tumor-associated proteases, would effectively crosslink andactivate the T cell or NK cell expressed co-stimulatory receptors in atumor-dependent manner to enhance the activity of T cells that areresponding to any tumor antigen via their endogenous T cell antigen orNK-activating receptors. The activation-dependent nature of these T cellor NK cell costimulatory receptors would focus the activity of theactivated multispecific activatable antibody to tumor-specific T cells,without activating all T cells independent of their antigen specificity.In one embodiment, at least the co-stimulatory receptor antibody of themultispecific activatable antibody is masked to prevent activation ofauto-reactive T cells that may be present in tissues that also expressthe antigen recognized by the tumor target-directed antibody in themultispecific activatable antibody, but whose activity is restricted bylack of co-receptor engagement.

One embodiment of the disclosure is a multispecific activatable antibodythat is activatable in a disease characterized by T celloverstimulation, such as, but not limited to, an autoimmune disease orinflammatory disease microenvironment. Such a multispecific activatableantibody includes an antibody, for example a IgG or scFv, directed to atarget comprising a surface antigen expressed in a tissue targeted by aT cell in autoimmune or inflammatory disease and an antibody, forexample a IgG or scFv, directed to an inhibitory receptor expressed onthe surface of a T cell or NK cell, wherein at least one of the diseasetissue target antibody and/or T cell inhibitory receptor antibody ismasked. Examples of inhibitory receptors include, but are not limitedto, BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs.Examples of a tissue antigen targeted by T cells in autoimmune diseaseinclude, but are not limited to, a surface antigen expressed on myelinor nerve cells in multiple sclerosis or a surface antigen expressed onpancreatic islet cells in Type 1 diabetes. In this embodiment, themultispecific activatable antibody when localized in the tissue underautoimmune attack or inflammation is activated and co-engages the T cellor NK cell inhibitory receptor to suppress the activity of autoreactiveT cells responding to any disease tissue-targeted antigens via theirendogenous TCR or activating receptors. In one embodiment, at least oneor multiple antibodies are masked to prevent suppression of T cellresponses in non-disease tissues where the target antigen may also beexpressed.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3 epsilon (CD3ε, also referred to herein asCD3e and CD3) scFv and a targeting antibody or antigen-binding fragmentthereof, where at least one of the anti-CD3ε scFv and/or the targetingantibody or antigen-binding portion thereof is masked. In someembodiments, the CD3ε scFv includes a first antibody or antigen-bindingfragment thereof (AB1) that binds CD3ε, where the AB1 is attached to amasking moiety (MM1) such that coupling of the MM1 reduces the abilityof the AB1 to bind CD3ε. In some embodiments, the targeting antibody orantigen-binding fragment thereof includes a second antibody or fragmentthereof that includes a second antibody or antigen-binding fragmentthereof (AB2) that binds a second target, where the AB2 is attached to amasking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second target. In some embodiments, the CD3ε scFvincludes a first antibody or antigen-binding fragment thereof (AB1) thatbinds CD3ε, where the AB1 is attached to a masking moiety (MM1) suchthat coupling of the MM1 reduces the ability of the AB1 to bind CD3ε,and the targeting antibody or antigen-binding fragment thereof includesa second antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3ε scFv and a cancer targeting antibody orantigen-binding fragment thereof, where at least one of the anti-CD3εscFv and/or the cancer targeting antibody or antigen-binding portionthereof is masked. In some embodiments, the CD3ε scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target. In some embodiments, theCD3ε scFv includes a first antibody or antigen-binding fragment thereof(AB1) that binds CD3ε, where the AB1 is attached to a masking moiety(MM1) such that coupling of the MM1 reduces the ability of the AB1 tobind CD3ε, and the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3ε scFv and a cancer targeting IgG antibodyor antigen-binding fragment thereof, where at least one of the anti-CD3εscFv and/or the cancer targeting IgG antibody or antigen-binding portionthereof is masked. In some embodiments, the CD3ε scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the CD3ε scFv includes a first antibody or antigen-bindingfragment thereof (AB1) that binds CD3ε, where the AB1 is attached to amasking moiety (MM1) such that coupling of the MM1 reduces the abilityof the AB1 to bind CD3ε, and the cancer targeting antibody IgG orantigen-binding fragment thereof includes a second antibody or fragmentthereof that includes a second antibody or antigen-binding fragmentthereof (AB2) that binds a second, cancer-related target, where the AB2is attached to a masking moiety (MM2) such that coupling of the MM2reduces the ability of the AB2 to bind the second, cancer-relatedtarget.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CD3 epsilon (CD3ε) scFv that is derived fromOKT3, where at least one of the targeting antibody or antigen-bindingfragment thereof and/or the OKT3 scFv or OKT3-derived scFv is masked. Insome embodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the targeting antibody or antigen-binding fragment thereofincludes a second antibody or fragment thereof that includes a secondantibody or antigen-binding fragment thereof (AB2) that binds a secondtarget, where the AB2 is attached to a masking moiety (MM2) such thatcoupling of the MM2 reduces the ability of the AB2 to bind the secondtarget. In some embodiments, the OKT3 scFv or OKT3-derived scFv includesa first antibody or antigen-binding fragment thereof (AB1) that bindsCD3ε, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CD3ε, and thetargeting antibody or antigen-binding fragment thereof includes a secondantibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an OKT3 scFv or OKT3-derived scFv and a cancertargeting antibody or antigen-binding fragment thereof, where at leastone of the OKT3 scFv or OKT3-derived scFv and/or the cancer targetingantibody or antigen-binding portion thereof is masked. In someembodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond, cancer-related target, where the AB2 is attached to a maskingmoiety (MM2) such that coupling of the MM2 reduces the ability of theAB2 to bind the second, cancer-related target. In some embodiments, theOKT3 scFv or OKT3-derived scFv includes a first antibody orantigen-binding fragment thereof (AB1) that binds CD3ε, where the AB1 isattached to a masking moiety (MM1) such that coupling of the MM1 reducesthe ability of the AB1 to bind CD3ε, and the cancer targeting antibodyor antigen-binding fragment thereof includes a second antibody orfragment thereof that includes a second antibody or antigen-bindingfragment thereof (AB2) that binds a second, cancer-related target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second, cancer-relatedtarget.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an OKT3 scFv or OKT3-derived scFv and a cancertargeting IgG antibody or antigen-binding fragment thereof, where atleast one of the OKT3 scFv or OKT3-derived scFv and/or the cancertargeting IgG antibody or antigen-binding portion thereof is masked. Insome embodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε. In someembodiments, the cancer targeting IgG antibody or antigen-bindingfragment thereof includes a second antibody or fragment thereof thatincludes a second antibody or antigen-binding fragment thereof (AB2)that binds a second, cancer-related target, where the AB2 is attached toa masking moiety (MM2) such that coupling of the MM2 reduces the abilityof the AB2 to bind the second, cancer-related target. In someembodiments, the OKT3 scFv or OKT3-derived scFv includes a firstantibody or antigen-binding fragment thereof (AB1) that binds CD3ε,where the AB1 is attached to a masking moiety (MM1) such that couplingof the MM1 reduces the ability of the AB1 to bind CD3ε, and the cancertargeting antibody IgG or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second,cancer-related target, where the AB2 is attached to a masking moiety(MM2) such that coupling of the MM2 reduces the ability of the AB2 tobind the second, cancer-related target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CTLA-4 scFv, where at least one of thetargeting antibody or antigen-binding fragment thereof and/or theanti-CTLA-4 scFv is masked. In some embodiments, the anti-CTLA-4 scFvincludes a first antibody or antigen-binding fragment thereof (AB1) thatbinds CTLA-4, where the AB1 is attached to a masking moiety (MM1) suchthat coupling of the MM1 reduces the ability of the AB1 to bind CTLA-4.In some embodiments, the targeting antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond target, where the AB2 is attached to a masking moiety (MM2) suchthat coupling of the MM2 reduces the ability of the AB2 to bind thesecond target. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4, andthe targeting antibody or antigen-binding fragment thereof includes asecond antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the T-cell engaging multispecific activatableantibody includes an anti-CTLA-4 scFv and a targeting IgG antibody orantigen-binding fragment thereof, where at least one of the anti-CTLA-4scFv and/or the targeting IgG antibody or antigen-binding portionthereof is masked. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4. Insome embodiments, the targeting IgG antibody or antigen-binding fragmentthereof includes a second antibody or fragment thereof that includes asecond antibody or antigen-binding fragment thereof (AB2) that binds asecond target, where the AB2 is attached to a masking moiety (MM2) suchthat coupling of the MM2 reduces the ability of the AB2 to bind thesecond target. In some embodiments, the anti-CTLA-4 scFv includes afirst antibody or antigen-binding fragment thereof (AB1) that bindsCTLA-4, where the AB1 is attached to a masking moiety (MM1) such thatcoupling of the MM1 reduces the ability of the AB1 to bind CTLA-4, andthe targeting antibody IgG or antigen-binding fragment thereof includesa second antibody or fragment thereof that includes a second antibody orantigen-binding fragment thereof (AB2) that binds a second target, wherethe AB2 is attached to a masking moiety (MM2) such that coupling of theMM2 reduces the ability of the AB2 to bind the second target.

In some embodiments, the multi-antigen targeting antibodies and/ormulti-antigen targeting activatable antibodies include at least a firstantibody or antigen-binding fragment thereof that binds a first targetand/or first epitope and a second antibody or antigen-binding fragmentthereof that binds a second target and/or a second epitope. In someembodiments, the multi-antigen targeting antibodies and/or multi-antigentargeting activatable antibodies bind two or more different targets. Insome embodiments, the multi-antigen targeting antibodies and/ormulti-antigen targeting activatable antibodies bind two or moredifferent epitopes on the same target. In some embodiments, themulti-antigen targeting antibodies and/or multi-antigen targetingactivatable antibodies bind a combination of two or more differenttargets and two or more different epitopes on the same target.

In some embodiments, a multispecific activatable antibody comprising anIgG has the IgG variable domains masked. In some embodiments, amultispecific activatable antibody comprising a scFv has the scFvdomains masked. In some embodiments, a multispecific activatableantibody has both IgG variable domains and scFv domains, where at leastone of the IgG variable domains is coupled to a masking moiety. In someembodiments, a multispecific activatable antibody has both IgG variabledomains and scFv domains, where at least one of the scFv domains iscoupled to a masking moiety. In some embodiments, a multispecificactivatable antibody has both IgG variable domains and scFv domains,where at least one of the IgG variable domains is coupled to a maskingmoiety and at least one of the scFv domains is coupled to a maskingmoiety. In some embodiments, a multispecific activatable antibody hasboth IgG variable domains and scFv domains, where each of the IgGvariable domains and the scFv domains is coupled to its own maskingmoiety. In some embodiments, one antibody domain of a multispecificactivatable antibody has specificity for a target antigen and anotherantibody domain has specificity for a T-cell surface antigen. In someembodiments, one antibody domain of a multispecific activatable antibodyhas specificity for a target antigen and another antibody domain hasspecificity for another target antigen. In some embodiments, oneantibody domain of a multispecific activatable antibody has specificityfor an epitope of a target antigen and another antibody domain hasspecificity for another epitope of the target antigen.

In a multispecific activatable antibody, a scFv can be fused to thecarboxyl terminus of the heavy chain of an IgG activatable antibody, tothe carboxyl terminus of the light chain of an IgG activatable antibody,or to the carboxyl termini of both the heavy and light chains of an IgGactivatable antibody. In a multispecific activatable antibody, a scFvcan be fused to the amino terminus of the heavy chain of an IgGactivatable antibody, to the amino terminus of the light chain of an IgGactivatable antibody, or to the amino termini of both the heavy andlight chains of an IgG activatable antibody. In a multispecificactivatable antibody, a scFv can be fused to any combination of one ormore carboxyl termini and one or more amino termini of an IgGactivatable antibody. In some embodiments, a masking moiety (MM) linkedto a cleavable moiety (CM) is attached to and masks an antigen bindingdomain of the IgG. In some embodiments, a masking moiety (MM) linked toa cleavable moiety (CM) is attached to and masks an antigen bindingdomain of at least one scFv. In some embodiments, a masking moiety (MM)linked to a cleavable moiety (CM) is attached to and masks an antigenbinding domain of an IgG and a masking moiety (MM) linked to a cleavablemoiety (CM) is attached to and masks an antigen binding domain of atleast one scFv.

The disclosure provides examples of multispecific activatable antibodystructures which include, but are not limited to, the following:(VL-CL)₂:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)₂;(VL-CL)₂:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM)₂;(MM-L1-CM-L2-VL-CL)₂: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*)₂;(MM-L1-CM-L2-VL-CL)₂: (VH-CH1-CH2-CH3-L4-VL*-L3-VH*)₂;(VL-CL)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL-CL)₂:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL-CL)₂:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂:(VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂:(VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)₂:(VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL)₂:(VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)2;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂;(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; or(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂,wherein: VL and VH represent the light and heavy variable domains of thefirst specificity, contained in the IgG; VL* and VH* represent thevariable domains of the second specificity, contained in the scFv; L1 isa linker peptide connecting the masking moiety (MM) and the cleavablemoiety (CM); L2 is a linker peptide connecting the cleavable moiety(CM), and the antibody; L3 is a linker peptide connecting the variabledomains of the scFv; L4 is a linker peptide connecting the antibody ofthe first specificity to the antibody of the second specificity; CL isthe light-chain constant domain; and CH1, CH2, CH3 are the heavy chainconstant domains. The first and second specificities may be toward anyantigen or epitope.

In some embodiments of a T-cell engaging multispecific activatableantibody, one antigen is typically an antigen present on the surface ofa tumor cell or other cell type associated with disease, such as, butnot limited to, any target listed in Table 1, such as, but not limitedto, EGFR, erbB2, EpCAM, Jagged, PD-L1, B7H3, or CD71 (transferrinreceptor), and another antigen is typically a stimulatory (also referredto herein as activating) or inhibitory receptor present on the surfaceof a T-cell, natural killer (NK) cell, myeloid mononuclear cell,macrophage, and/or other immune effector cell, such as, but not limitedto, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56,CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3,NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain conferringspecificity to the T-cell surface antigen may also be substituted by aligand or ligand domain that binds to a T-cell receptor, a NK-cellreceptor, a macrophage receptor, and/or other immune effector cellreceptor, such as, but not limited to, B7-1, B7-2, B7H3, PD-L1, PD-L2,or TNFSF9. In some embodiments of a multi-antigen targeting activatableantibody, one antigen is selected from the group of targets listed inTable 1, and another antigen is selected from the group of targetslisted in Table 1.

In some embodiments, the targeting antibody is an anti-EGFR antibody. Insome embodiments, the targeting antibody is C225v5, which is specificfor binding to EGFR. In some embodiments, the targeting antibody isC225, which is specific for binding to EGFR. In some embodiments, thetargeting antibody is C225v4, which is specific for binding to EGFR. Insome embodiments, the targeting antibody is C225v6, which is specificfor binding to EGFR. In some embodiments, the targeting antibody is ananti-Jagged antibody. In some embodiments, the targeting antibody is4D11, which is specific for binding to human and mouse Jagged 1 andJagged 2. In some embodiments, the targeting antibody is 4D11v2, whichis specific for binding to human and mouse Jagged 1 and Jagged 2.

In some embodiments, the targeting antibody can be in the form anactivatable antibody. In some embodiments, the scFv(s) can be in theform of a Pro-scFv (see, e.g., WO 2009/025846, WO 2010/081173).

In some embodiments, the scFv is specific for binding CDR3ε, and is oris derived from an antibody or fragment thereof that binds CDR3ε, e.g.,CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, thescFv is specific for binding CTLA-4 (also referred to herein as CTLA andCTLA4).

In some embodiments, the anti-CTLA-4 scFv includes the amino acidsequence:

(SEQ ID NO: 692) GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSAS

In some embodiments, the anti-CTLA-4 scFv includes the amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 692.

In some embodiments, the anti-CDR scFv includes the amino acid sequence:

(SEQ ID NO: 693) GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSN PFTFGSGTKLEINR

In some embodiments, the anti-CDR scFv includes the amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 693.

In some embodiments, the scFv is specific for binding one or moreT-cells, one or more NK-cells and/or one or more macrophages. In someembodiments, the scFv is specific for binding a target selected from thegroup consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28,CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1,TIGIT, TIM3, or VISTA.

In some embodiments, the multispecific activatable antibody alsoincludes an agent conjugated to the AB. In some embodiments, the agentis a therapeutic agent. In some embodiments, the agent is anantineoplastic agent. In some embodiments, the agent is a toxin orfragment thereof. In some embodiments, the agent is conjugated to themultispecific activatable antibody via a linker. In some embodiments,the agent is conjugated to the AB via a cleavable linker. In someembodiments, the agent is conjugated to the AB via a linker thatincludes at least one uPA-cleavable substrate sequence or at least onematriptase-cleavable substrate sequence. In some embodiments, the linkeris a non-cleavable linker. In some embodiments, the agent is amicrotubule inhibitor. In some embodiments, the agent is a nucleic aciddamaging agent, such as a DNA alkylator or DNA intercalator, or otherDNA damaging agent. In some embodiments, the linker is a cleavablelinker. In some embodiments, the agent is an agent selected from thegroup listed in Table 4. In some embodiments, the agent is a dolastatin.In some embodiments, the agent is an auristatin or derivative thereof.In some embodiments, the agent is auristatin E or a derivative thereof.In some embodiments, the agent is monomethyl auristatin E (MMAE). Insome embodiments, the agent is monomethyl auristatin D (MMAD). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof. In some embodiments, theagent is a pyrrolobenzodiazepine.

In some embodiments, the multispecific activatable antibody alsoincludes a detectable moiety. In some embodiments, the detectable moietyis a diagnostic agent.

In some embodiments, the multispecific activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, themultispecific activatable antibody can be engineered to include one ormore disulfide bonds.

The disclosure also provides an isolated nucleic acid molecule encodinga multispecific activatable antibody described herein, as well asvectors that include these isolated nucleic acid sequences. Thedisclosure provides methods of producing a multispecific activatableantibody by culturing a cell under conditions that lead to expression ofthe activatable antibody, wherein the cell comprises such a nucleic acidmolecule. In some embodiments, the cell comprises such a vector.

The disclosure also provides a method of manufacturing multispecificactivatable antibodies of the disclosure by (a) culturing a cellcomprising a nucleic acid construct that encodes the multispecificactivatable antibody under conditions that lead to expression of themultispecific activatable, and (b) recovering the multispecificactivatable antibody.

The disclosure also provides multispecific activatable antibodies and/ormultispecific activatable antibody compositions that include at least afirst antibody or antigen-binding fragment thereof (AB1) thatspecifically binds a first target or first epitope and a second antibodyor antigen-biding fragment thereof (AB2) that binds a second target or asecond epitope, where at least AB1 is coupled or otherwise attached to amasking moiety (MM1), such that coupling of the MM1 reduces the abilityof AB1 to bind its target. In some embodiments, the MM1 is coupled toAB1 via a first cleavable moiety (CM1) sequence that includes asubstrate for a protease, for example, a protease that is co-localizedwith the target of AB1 at a treatment site or a diagnostic site in asubject. The multispecific activatable antibodies provided herein arestable in circulation, activated at intended sites of therapy and/ordiagnosis but not in normal, i.e., healthy tissue, and, when activated,exhibit binding to the target of AB1 that is at least comparable to thecorresponding, unmodified multispecific antibody.

In some embodiments, the multispecific activatable antibody comprises alinking peptide between the MM1 and the CM1.

In some embodiments, the multispecific activatable antibody comprises alinking peptide between the CM1 and the AB1.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), and at least a portionof the multispecific activatable antibody has the structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1. In some embodiments, the twolinking peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 385) and (GGGS)_(n) (SEQ ID NO: 386), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 387), GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO:389), GSGGG (SEQ ID NO: 390), GGGSG (SEQ ID NO: 391), and GSSSG (SEQ IDNO: 392).

In some embodiments, the multispecific activatable antibody includes atleast a first antibody or antigen-binding fragment thereof (AB1) thatspecifically binds a first target or first epitope and a second antibodyor antigen-binding fragment thereof (AB2) that specifically binds asecond target or second epitope. In some embodiments, each of the AB inthe multispecific activatable antibody is independently selected fromthe group consisting of a monoclonal antibody, domain antibody, singlechain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a singledomain heavy chain antibody, and a single domain light chain antibody.In some embodiments, each of the AB in the multispecific activatableantibody is a rodent (e.g., mouse or rat), chimeric, humanized or fullyhuman monoclonal antibody.

In some embodiments, each of the AB in the multispecific activatableantibody has an equilibrium dissociation constant of about 100 nM orless for binding to its corresponding target or epitope.

In some embodiments, MM1 has an equilibrium dissociation constant forbinding to its corresponding AB that is greater than the equilibriumdissociation constant of the AB to its corresponding target or epitope.

In some embodiments, MM1 has an equilibrium dissociation constant forbinding to its corresponding AB that is no more than the equilibriumdissociation constant of the AB to its corresponding target or epitope.

In some embodiments, MM1 does not interfere or compete with itscorresponding AB for binding to the corresponding target or epitope whenthe multispecific activatable antibody is in a cleaved state.

In some embodiments, MM1 is a polypeptide of about 2 to 40 amino acidsin length. In some embodiments, each of the MM in the multispecificactivatable antibody is a polypeptide of no more than 40 amino acids inlength.

In some embodiments, MM1 has a polypeptide sequence that is differentfrom that of target of the corresponding AB.

In some embodiments, MM1 has a polypeptide sequence that is no more than50% identical to any natural binding partner of the corresponding AB. Insome embodiments, MM1 has a polypeptide sequence that is no more than25% identical to any natural binding partner of the corresponding AB. Insome embodiments, MM1 has a polypeptide sequence that is no more than10% identical to any natural binding partner of the corresponding AB.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 40 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 100 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 1000 times greater thanthe K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, the coupling of MM1 reduces the ability of thecorresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM1 towardsits corresponding target or epitope is at least 10,000 times greaterthan the K_(d) of the AB when not coupled to the MM1 towards itscorresponding target or epitope.

In some embodiments, MM1 is an amino acid sequence selected from a MMdisclosed herein.

In some embodiments, the multispecific activatable antibody includes atleast a second masking moiety (MM2) that inhibits the binding of the AB2to its target when the multispecific activatable antibody is in anuncleaved state, and a second cleavable moiety (CM2) coupled to the AB2,wherein the CM2 is a polypeptide that functions as a substrate for asecond protease. In some embodiments, CM2 is a polypeptide of no morethan 15 amino acids long. In some embodiments, the second protease isco-localized with the second target or epitope in a tissue, and whereinthe second protease cleaves the CM2 in the multispecific activatableantibody when the multispecific activatable antibody is exposed to thesecond protease. In some embodiments, the first protease and the secondprotease are co-localized with the first target or epitope and thesecond target or epitope in a tissue. In some embodiments, the firstprotease and the second protease are the same protease. In someembodiments, CM1 and CM2 are different substrates for the same protease.In some embodiments, the protease is selected from the group consistingof those shown in Table 7. In some embodiments, the first protease andthe second protease are different proteases. In some embodiments, thefirst protease and the second protease are different proteases selectedfrom the group consisting of those shown in Table 7.

In some embodiments, each of the MM in the multispecific activatableantibody, e.g., MM1 and at least MM2, has an equilibrium dissociationconstant for binding to its corresponding AB that is greater than theequilibrium dissociation constant of the AB to its corresponding targetor epitope.

In some embodiments, each of the MM in the multispecific activatableantibody has an equilibrium dissociation constant for binding to itscorresponding AB that is no more than the equilibrium dissociationconstant of the AB to its corresponding target or epitope.

In some embodiments, each of the MM in the multispecific activatableantibody does not interfere or compete with its corresponding AB forbinding to the corresponding target or epitope when the multispecificactivatable antibody is in a cleaved state.

In some embodiments, each of the MM1 in the multispecific activatableantibody is a polypeptide of about 2 to 40 amino acids in length. Insome embodiments, each of the MM in the multispecific activatableantibody is a polypeptide of no more than 40 amino acids in length.

In some embodiments, each of the MM in the multispecific activatableantibody has a polypeptide sequence that is different from that oftarget of the corresponding AB.

In some embodiments, each of the MM in the multispecific activatableantibody has a polypeptide sequence that is no more than 50% identicalto any natural binding partner of the corresponding AB. In someembodiments, each of the MM in the multispecific activatable antibodyhas a polypeptide sequence that is no more than 25% identical to anynatural binding partner of the corresponding AB. In some embodiments,each of the MM1 in the multispecific activatable antibody has apolypeptide sequence that is no more than 10% identical to any naturalbinding partner of the corresponding AB.

In some embodiments, the coupling of each of the MM1 reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 40 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 100 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 1000 times greater thanthe K_(d) of the AB when not coupled to the MM towards its correspondingtarget or epitope.

In some embodiments, the coupling of each of the MM reduces the abilityof the corresponding AB to bind its target or epitope such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsits corresponding target or epitope is at least 10,000 times greaterthan the K_(d) of the AB when not coupled to the MM towards itscorresponding target or epitope.

In some embodiments, each of the MM is an amino acid sequence selectedfrom a MM disclosed herein.

In some embodiments, at least one of CM1 and/or CM2 is cleaved by atleast one protease selected from uPA and matriptase. In someembodiments, at least one of CM1 and/or CM2 includes an amino acidsequence selected from the group consisting of a core CM consensussequence shown in Tables 8A-8J, a subgenus of a core CM consensussequence shown in Tables 8A-8J, an expanded consensus sequence based onone of the core CM consensus sequence shown in Tables 8A-8J, an. In someembodiments, the expanded consensus sequence is a consensus sequenceshown in Tables 9A-9J-3, a core CM consensus sequence shown in Tables10A-10D, a subgenus of a core CM consensus sequence shown in Tables10A-10D, and a consensus sequence shown in Tables 11A-11D.

In some embodiments, at least one of CM1 and/or CM2 includes an aminoacid sequence selected from the group consisting of SEQ ID NOs: 163-267.

In some embodiments, the protease that cleaves the first cleavablemoiety (CM1) sequence is co-localized with the target of the AB1 in themultispecific activatable antibody in a tissue, and the protease cleavesthe CM1 in the multispecific activatable antibody when the multispecificactivatable antibody is exposed to the protease.

In some embodiments, the multispecific activatable antibody includesmore than one cleavable moiety sequence, and the protease that cleavesat least one cleavable moiety sequence is co-localized with the targetof at least one of the AB regions in the multispecific activatableantibody in a tissue, and the protease cleaves the CM in themultispecific activatable antibody when the multispecific activatableantibody is exposed to the protease.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least twofold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least threefold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least fourfold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least fivefold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least tenfold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM, e.g., CM1 and at least CM2, is positionedin the multispecific activatable antibody such that in the uncleavedstate, binding of the multispecific activatable antibody to a target ofone of the AB regions is reduced to occur with an equilibriumdissociation constant that is at least 20-fold greater than theequilibrium dissociation constant of an unmodified AB binding to itstarget, and whereas in the cleaved state, the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 40-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 50-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 100-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM is positioned in the multispecificactivatable antibody such that in the uncleaved state, binding of themultispecific activatable antibody to a target of one of the AB regionsis reduced to occur with an equilibrium dissociation constant that is atleast 200-fold greater than the equilibrium dissociation constant of anunmodified AB binding to its target, and whereas in the cleaved state,the AB binds its target.

In some embodiments, each CM in the multispecific activatable antibodyis a polypeptide of up to 15 amino acids in length.

In some embodiments, at least one CM in the multispecific activatableantibody includes an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 163-267 and the other CM includes the aminoacid sequence LSGRSDNH (SEQ ID NO: 406). In some embodiments, at leastone CM includes the amino acid sequence LSGRSDNH (SEQ ID NO: 406). Insome embodiments, at least one cleavable moiety is selected for use witha specific protease, for example a protease that is known to beco-localized with at least one target of the multispecific activatableantibody. For example, suitable cleavable moieties for use in themultispecific activatable antibodies of the disclosure are cleaved by atleast a protease such as urokinase, legumain, and/or matriptase (alsoreferred to herein as MT-SP1 or MTSP1). In some embodiments, a suitablecleavable moiety includes at least one amino acid sequence selected fromthe group consisting of SEQ ID NO: 163-267.

In some embodiments, one CM is a substrate for at least one proteaseselected from uPA and matriptase, and the other CM in the multispecificactivatable antibody is a substrate for a protease selected from thegroup consisting of those shown in Table 7. In some embodiments, theprotease is selected from the group consisting of uPA, legumain,matriptase, ADAM17, BMP-1, TMPRSS3, TMPRSS4, neutrophil elastase, MMP-7,MMP-9, MMP-12, MMP-13, and MMP-14. In some embodiments, the protease isa cathepsin, such as, but not limited to, cathepsin S. In someembodiments, each CM in the multispecific activatable antibody is asubstrate for a protease selected from the group consisting of uPA(urokinase plasminogen activator), legumain and matriptase. In someembodiments, the protease comprises uPA. In some embodiments, theprotease comprises legumain. In some embodiments, the protease comprisesmatriptase.

In some embodiments, at least one CM in the multispecific activatableantibody is a substrate for at least two proteases. In some embodiments,at least one CM in the multispecific activatable antibody is a substratefor at least two proteases, wherein one of the proteases is selectedfrom the group consisting of uPA and matriptase and the other proteaseis selected from the group consisting of those shown in Table 7. In someembodiments, at least one CM in the multispecific activatable antibodyis a substrate for at least two proteases selected from the groupconsisting of uPA, legumain and matriptase.

In some embodiments, the multispecific activatable antibody includes atleast a first CM (CM1) and a second CM (CM2). In some embodiments, CM1and CM2 are part of a single cleavable linker that joins an MM to an AB.In some embodiments, CM1 is part of a cleavable linker that joins MM1 toAB1, and CM2 is part of a separate cleavable linker that joins an MM2 toAB2. In some embodiments, a multispecific activatable antibody comprisesmore than two CMs. In some embodiments, such a multispecific activatableantibody comprises more than two CMs and more than two MMs. In someembodiments, CM1 and CM2 are each polypeptides of no more than 15 aminoacids long. In some embodiments, at least one of the first CM and thesecond CM is a polypeptide that functions as a substrate for a proteaseselected from the group consisting of those listed in Table 7. In someembodiments, at least one of the first CM and the second CM is apolypeptide that functions as a substrate for a protease selected fromthe group consisting of uPA, legumain, and matriptase. In someembodiments, the first CM is cleaved by a first cleaving agent selectedfrom the group consisting of uPA, legumain, and matriptase in a targettissue and the second CM is cleaved by a second cleaving agent in atarget tissue. In some embodiments, the other protease is selected fromthe group consisting of those shown in Table 7. In some embodiments, thefirst cleaving agent and the second cleaving agent are the same proteaseselected from the group consisting of those listed in Table 7, and thefirst CM and the second CM are different substrates for the enzyme. Insome embodiments, the first cleaving agent and the second cleaving agentare the same protease selected from the group consisting of uPA,legumain, and matriptase, and the first CM and the second CM aredifferent substrates for the enzyme. In some embodiments, the firstcleaving agent and the second cleaving agent are the same proteaseselected from the group listed in Table 7, and the first CM and thesecond CM are the same substrate. In some embodiments, the firstcleaving agent and the second cleaving agent are different proteases. Insome embodiments, the first cleaving agent and the second cleaving agentare different proteases selected from the group consisting of thoseshown in Table 7. In some embodiments, the first cleaving agent and thesecond cleaving agent are co-localized in the target tissue. In someembodiments, the first CM and the second CM are cleaved by at least onecleaving agent in the target tissue.

In some embodiments, the multispecific activatable antibody is exposedto and cleaved by a protease such that, in the activated or cleavedstate, the activated multispecific activatable antibody includes a lightchain amino acid sequence that includes at least a portion of LP2 and/orCM sequence after the protease has cleaved the CM.

The disclosure also provides compositions and methods that include amultispecific activatable antibody that includes at least a firstantibody or antibody fragment (AB1) that specifically binds a target anda second antibody or antibody fragment (AB2), where at least the firstAB in the multispecific activatable antibody is coupled to a maskingmoiety (MM1) that decreases the ability of AB1 to bind its target. Insome embodiments, each AB is coupled to a MM that decreases the abilityof its corresponding AB to each target. For example, in bispecificactivatable antibody embodiments, AB1 is coupled to a first maskingmoiety (MM1) that decreases the ability of AB1 to bind its target, andAB2 is coupled to a second masking moiety (MM2) that decreases theability of AB2 to bind its target. In some embodiments, themultispecific activatable antibody comprises more than two AB regions;in such embodiments, AB1 is coupled to a first masking moiety (MM1) thatdecreases the ability of AB1 to bind its target, AB2 is coupled to asecond masking moiety (MM2) that decreases the ability of AB2 to bindits target, AB3 is coupled to a third masking moiety (MM3) thatdecreases the ability of AB3 to bind its target, and so on for each ABin the multispecific activatable antibody.

In some embodiments, the multispecific activatable antibody furtherincludes at least one cleavable moiety (CM) that is a substrate for aprotease, where the CM links a MM to an AB. For example, in someembodiments, the multispecific activatable antibody includes at least afirst antibody or antibody fragment (AB1) that specifically binds atarget and a second antibody or antibody fragment (AB2), where at leastthe first AB in the multispecific activatable antibody is coupled via afirst cleavable moiety (CM1) to a masking moiety (MM1) that decreasesthe ability of AB1 to bind its target. In some bispecific activatableantibody embodiments, AB1 is coupled via CM1 to MM1, and AB2 is coupledvia a second cleavable moiety (CM2) to a second masking moiety (MM2)that decreases the ability of AB2 to bind its target. In someembodiments, the multispecific activatable antibody comprises more thantwo AB regions; in some of these embodiments, AB1 is coupled via CM1 toMM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled via a thirdcleavable moiety (CM3) to a third masking moiety (MM3) that decreasesthe ability of AB3 to bind its target, and so on for each AB in themultispecific activatable antibody.

Activatable Antibodies Having Non-Binding Steric Moieties or BindingPartners for Non-Binding Steric Moieties

The disclosure also provides activatable antibodies that includenon-binding steric moieties (NB) or binding partners (BP) fornon-binding steric moieties, where the BP recruits or otherwise attractsthe NB to the activatable antibody. The activatable antibodies providedherein include, for example, an activatable antibody that includes anon-binding steric moiety (NB), a cleavable linker (CL) and antibody orantibody fragment (AB) that binds a target; an activatable antibody thatincludes a binding partner for a non-binding steric moiety (BP), a CLand an AB; and an activatable antibody that includes a BP to which an NBhas been recruited, a CL and an AB that binds the target. Activatableantibodies in which the NB is covalently linked to the CL and AB of theactivatable antibody or is associated by interaction with a BP that iscovalently linked to the CL and AB of the activatable antibody arereferred to herein as “NB-containing activatable antibodies.” Byactivatable or switchable is meant that the activatable antibodyexhibits a first level of binding to a target when the activatableantibody is in an inhibited, masked or uncleaved state (i.e., a firstconformation), and a second level of binding to the target when theactivatable antibody is in an uninhibited, unmasked and/or cleaved state(i.e., a second conformation, i.e., activated antibody), where thesecond level of target binding is greater than the first level of targetbinding. The activatable antibody compositions can exhibit increasedbioavailability and more favorable biodistribution compared toconventional antibody therapeutics.

In some embodiments, activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe at non-treatment sites and/or non-diagnostic sites if the AB werenot masked or otherwise inhibited from binding to such a site.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein the NB is apolypeptide that does not bind specifically to the AB; the CL is apolypeptide that includes a substrate (S) for an enzyme; the CL ispositioned such that in an uncleaved state, the NB interferes withbinding of the AB to the target and in a cleaved state, the NB does notinterfere with binding of the AB to the target; and the NB does notinhibit cleavage of the CL by the enzyme. As used herein and throughout,the term polypeptide refers to any polypeptide that includes at leasttwo amino acid residues, including larger polypeptides, full-lengthproteins and fragments thereof, and the term polypeptide is not limitedto single-chain polypeptides and can include multi-unit, e.g.,multi-chain, polypeptides. In cases where the polypeptide is of ashorter length, for example, less than 50 amino acids total, the termspeptide and polypeptide are used interchangeably herein, and in caseswhere the polypeptide is of a longer length, e.g., 50 amino acids orgreater, the terms polypeptide and protein are used interchangeablyherein.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)CL is a polypeptide of up to 50 amino acids in length that includes asubstrate (S) for an enzyme, e.g., a protease selected from matriptaseand uPA; (iii) the CL is positioned such that in an uncleaved state, theNB interferes with binding of the AB to the target and in a cleavedstate, the NB does not interfere with binding of the AB to the target;and (iv) the NB does not inhibit cleavage of the CL by the enzyme. Forexample, the CL has a length of up to 15 amino acids, a length of up to20 amino acids, a length of up to 25 amino acids, a length of up to 30amino acids, a length of up to 35 amino acids, a length of up to 40amino acids, a length of up to 45 amino acids, a length of up to 50amino acids, a length in the range of 10-50 amino acids, a length in therange of 15-50 amino acids, a length in the range of 20-50 amino acids,a length in the range of 25-50 amino acids, a length in the range of30-50 amino acids, a length in the range of 35-50 amino acids, a lengthin the range of 40-50 amino acids, a length in the range of 45-50 aminoacids, a length in the range of 10-40 amino acids, a length in the rangeof 15-40 amino acids, a length in the range of 20-40 amino acids, alength in the range of 25-40 amino acids, a length in the range of 30-40amino acids, a length in the range of 35-40 amino acids, a length in therange of 10-30 amino acids, a length in the range of 15-30 amino acids,a length in the range of 20-30 amino acids, a length in the range of25-30 amino acids, a length in the range of 10-20 amino acids, or alength in the range of 10-15 amino acids.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)the CL is a polypeptide that includes a substrate (S) for an enzyme,e.g., a protease selected from matriptase and uPA; (iii) the CL ispositioned such that in an uncleaved state, the NB interferes withbinding of the AB to the target and in a cleaved state, the NB does notinterfere with binding of the AB to the target; (iv) the NB does notinhibit cleavage of the CL by the enzyme; and (v) the activatableantibody has the structural arrangement from N-terminus to C-terminus asfollows in the uncleaved state: NB-CL-AB or AB-CL-NB.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to the target, wherein (i) the NBincludes a polypeptide that does not bind specifically to the AB; (ii)the CL is a polypeptide that includes a substrate (S) for an enzyme,e.g., a protease selected from matriptase and uPA; (iii) the CL ispositioned such that in an uncleaved state, the NB interferes withbinding of the AB to the target and in a cleaved state, the NB does notinterfere with binding of the AB to the target, and wherein the NB inthe uncleaved activatable antibody reduces the ability of the AB to bindthe target by at least 50%, for example, by at least 60%, by at least70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%,by at least 95%, by at least 96%, by at least 97%, by at least 98%, byat least 99%, by at least 100% as compared to the ability of the cleavedAB to bind the target; and (iv) the NB does not inhibit cleavage of theCL by the enzyme. The reduction in the ability of the AB to bind thetarget is determined, e.g., using an assay as described herein or an invitro target displacement assay such as, for example, the assaydescribed in PCT Publication Nos. WO 2009/025846 and WO 2010/081173.

In one embodiment, the activatable antibody includes a binding partner(BP) for a non-binding steric moiety (NB); a cleavable linker (CL); andan antibody or antibody fragment (AB) that binds specifically to thetarget, wherein the BP is a polypeptide that binds to the NB whenexposed thereto; the NB does not bind specifically to the AB; the CL isa polypeptide that includes a substrate (S) for an enzyme, e.g., aprotease selected from matriptase and uPA; the CL is positioned suchthat in an uncleaved state in the presence of the NB, the NB interfereswith binding of the AB to the target and in a cleaved state, the NB doesnot interfere with binding of the AB to the target and the BP does notinterfere with binding of the AB to the target; and the NB and the BP donot inhibit cleavage of the CL by the enzyme. In some examples of thisembodiment, the BP of the activatable antibody is optionally bound tothe NB. In one embodiment, the NB is recruited by the BP of theactivatable antibody in vivo.

In some examples of any of these activatable antibody embodiments, theactivatable antibody is formulated as a composition. In some of theseembodiments, the composition also includes the NB, where the NB isco-formulated with the activatable antibody that includes the BP, theCL, and the AB. In some examples of this embodiment, the BP is selectedfrom the group consisting of an albumin binding peptide, a fibrinogenbinding peptide, a fibronectin binding peptide, a hemoglobin bindingpeptide, a transferrin binding peptide, an immunoglobulin domain bindingpeptide, and other serum protein binding peptides.

In some examples of any of these activatable antibody embodiments, theNB is a soluble, globular protein. In some examples of any of theseactivatable antibody embodiments, the NB is a protein that circulates inthe bloodstream. In some examples of any of these activatable antibodyembodiments, the NB is selected from the group consisting of albumin,fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulindomain, and other serum proteins.

In some examples of any of these activatable antibody embodiments, theCL is a polypeptide that includes a substrate (S) for a proteaseselected from matriptase and uPA. In some examples of any of theseactivatable antibody embodiments, the protease is co-localized with thein a tissue, and the protease cleaves the CL in the activatable antibodywhen the activatable antibody is exposed to the protease. In someexamples of any of these activatable antibody embodiments, the CL is apolypeptide of up to 50 amino acids in length. In some examples of anyof these activatable antibody embodiments, the CL is a polypeptide thatincludes a substrate (S) having a length of up to 15 amino acids, e.g.,3 amino acids long, 4 amino acids long, 5 amino acids long, 6 aminoacids long, 7 amino acids long, 8 amino acids long, 9 amino acids long,10 amino acids long, 11 amino acids long, 12 amino acids long, 13 aminoacids long, 14 amino acids long, or 15 amino acids long.

In some examples of any of these activatable antibody embodiments, theactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: NB-CL-AB, AB-CL-NB,BP-CL-AB or AB-CL-BP. In embodiments where the activatable antibodyincludes a BP and the activatable antibody is in the presence of thecorresponding NB, the activatable antibody has a structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:NB:BP-CM-AB or AB-CM-BP:NB, where “:” represents an interaction, e.g.,binding, between the NB and BP.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes an antibody or antigen-binding fragmentthereof that specifically binds a given target and is a monoclonalantibody, domain antibody, single chain, Fab fragment, a F(ab′)₂fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody,or a single domain light chain antibody. In some embodiments, such anantibody or immunologically active fragment thereof that binds thetarget a mouse, other rodent, chimeric, humanized or fully humanmonoclonal antibody.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes a combination of a variable heavy chainregion comprising an amino acid sequence presented herein and a variablelight chain region comprising an amino acid sequence presented herein.In some embodiments, the activatable antibody includes a combination ofa variable heavy chain region comprising an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to an amino acid sequence presented herein, and a variablelight chain region comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence presented herein.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes an agent conjugated to the AB. Insome embodiments, the agent is a therapeutic agent. In some embodiments,the agent is an antineoplastic agent. In some embodiments, the agent isa toxin or fragment thereof. In some embodiments, the agent isconjugated to the AB via a linker. In some embodiments, the linker is acleavable linker. In some embodiments, the agent is conjugated to the ABvia a noncleavable linker. In some embodiments, the agent is an agentselected from the group listed in Table 3. In some embodiments, theagent is a microtubule inhibitor. In some embodiments, the agent is anucleic acid damaging agent, such as a DNA alkylator or DNAintercalator, or other DNA damaging agent. In some embodiments, theagent is a dolastatin. In some embodiments, the agent is an auristatinor derivative thereof. In some embodiments, the agent is auristatin E ora derivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes a detectable moiety. In someembodiments, the detectable moiety is a diagnostic agent.

In some examples of any of these activatable antibody embodiments, theactivatable antibody also includes a spacer. In some examples of any ofthese activatable antibody embodiments, the activatable antibody alsoincludes a signal peptide. In some embodiments, the signal peptide isconjugated to the activatable antibody via a spacer. In some examples ofany of these activatable antibody embodiments, the spacer is joineddirectly to the MM of the activatable antibody.

In some embodiments, the serum half-life of the activatable antibody islonger than that of the corresponding antibody; e.g., the pK of theactivatable antibody is longer than that of the corresponding antibody.In some embodiments, the serum half-life of the activatable antibody issimilar to that of the corresponding antibody. In some embodiments, theserum half-life of the activatable antibody is at least 15 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 11 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 9 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 8 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 7 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 days when administered to an organism. In some examples of any ofthese activatable antibody embodiments, the serum half-life of theactivatable antibody is at least 5 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 4 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least3 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 2 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 24 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 20 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least18 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 16 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 14 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 12 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 8 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 6 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 4 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least3 hours when administered to an organism.

The disclosure also provides an isolated nucleic acid molecule encodingany of these activatable antibodies, as well as vectors that includethese isolated nucleic acid sequences. The disclosure provides methodsof producing an activatable antibody by culturing a cell underconditions that lead to expression of the activatable antibody, whereinthe cell comprises such a nucleic acid sequence. In some embodiments,the cell comprises such a vector.

The dissociation constant (K_(d)) of the NB-containing activatableantibody toward the target is greater than the K_(d) of the AB towardsthe target when it is not associated with the NB or NB:BP. Thedissociation constant (K_(d)) of the NB-containing activatable antibodytoward the target is greater than the K_(d) of the parental AB towardsthe target. For example, the K_(d) of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times greater than the K_(d) of the AB when it is notassociated with the NB or NB:BP or the K_(d) of the parental AB towardsthe target. Conversely, the binding affinity of the NB-containingactivatable antibody towards the target is lower than the bindingaffinity of the AB when it is not associated with the NB or NB:BP orlower than the binding affinity of the parental AB towards the target.For example, the binding affinity of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB whenit is not associated with the NB or NB:BP or lower than the bindingaffinity of the parental AB towards the target.

When the NB-containing activatable antibody is in the presence of thetarget, specific binding of the AB to the target is reduced orinhibited, as compared to the specific binding of the AB when it is notassociated with the NB or NB:BP. When the NB-containing activatableantibody is in the presence of the target, specific binding of the AB tothe target is reduced or inhibited, as compared to the specific bindingof the parental AB to the target. When compared to the binding of the ABnot associated with an NB or NB:BP or the binding of the parental AB tothe target, the ability of the NB-containing activatable antibody tobind the target is reduced, for example, by at least 50%, 60%, 70%, 80%,90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at least2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10,15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months or longer when measured in vitro and/or in vivo.

When the NB-containing activatable antibody is in the presence of thetarget but not in the presence of a modifying agent (for example aprotease or other enzyme), specific binding of the AB to the target isreduced or inhibited, as compared to the specific binding of the AB whenit is not associated with the NB or NB:BP. When the NB-containingactivatable antibody is in the presence of the target but not in thepresence of a modifying agent (for example a protease, other enzyme,reduction agent, or light), specific binding of the AB to the target isreduced or inhibited, as compared to the specific binding of theparental AB to the target. When compared to the binding of the AB notassociated with an NB or NB:BP or the binding of the parental AB to thetarget, the ability of the NB-containing activatable antibody to bindthe target is reduced, for example, by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months or longer when measured in vitro and/or in vivo.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes an agent conjugated to the AB to producean activatable antibody conjugate. In some embodiments of theactivatable antibody conjugate, the agent is a therapeutic agent. Insome embodiments, the agent is a diagnostic agent. In some embodiments,the agent is a detectable marker. In some embodiments of the activatableantibody conjugate, the agent is an antineoplastic agent. In someembodiments of the activatable antibody conjugate, the agent is a toxinor fragment thereof. In some embodiments of the activatable antibodyconjugate, the agent is conjugated to the AB via a linker. In someembodiments of the activatable antibody conjugate, the linker is acleavable linker. In some embodiments, the agent is conjugated to the ABvia a noncleavable linker. In some embodiments, the agent is amicrotubule inhibitor. In some embodiments, the agent is a nucleic aciddamaging agent, such as a DNA alkylator or DNA intercalator, or otherDNA damaging agent. In some embodiments, the agent is an agent selectedfrom the group listed in Table 3. In some embodiments, the agent is adolastatin. In some embodiments, the agent is an auristatin orderivative thereof. In some embodiments, the agent is auristatin E or aderivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is monomethylauristatin D (MMAD). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.In some embodiments, the agent is a pyrrolobenzodiazepine.

In some examples of any of these activatable antibody embodiments, theactivatable antibodies are dual-target binding activatable antibodies.Such dual target binding activatable antibodies contain two Abs that maybind the same or different targets. In specific embodiments,dual-targeting activatable antibodies contain bispecific antibodies orantibody fragments.

Dual target binding activatable antibodies are designed so as to have aCL cleavable by a cleaving agent, e.g., a protease selected frommatriptase and uPA, that is co-localized in a target tissue with one orboth of the targets capable of binding to the ABs of the activatableantibodies. Dual target binding activatable antibodies with more thanone AB to the same or different targets can be designed so as to havemore than one CL, wherein the first CL is cleavable by a cleaving agentin a first target tissue and wherein the second CL is cleavable by acleaving agent in a second target tissue, with one or more of thetargets binding to the ABs of the activatable antibodies. In oneembodiment, the first and second target tissues are spatially separated,for example, at different sites in the organism. In one embodiment, thefirst and second target tissues are the same tissue temporallyseparated, for example the same tissue at two different points in time,for example the first time point is when the tissue is an early stagetumor, and the second time point is when the tissue is a late stagetumor.

The disclosure also provides nucleic acid molecules encoding theactivatable antibodies described herein. The disclosure also providesvectors that include these nucleic acids. The activatable antibodiesdescribed herein are produced by culturing a cell under conditions thatlead to expression of the activatable antibody, wherein the cellincludes these nucleic acid molecules or vectors.

The disclosure also provides methods of manufacturing activatableantibodies. In one embodiment, the method includes the steps of (a)culturing a cell that includes a nucleic acid construct that encodes theactivatable antibody under conditions that lead to expression of theactivatable antibody, wherein the activatable antibody includes (i) anon-binding steric moiety (NB); (ii) a cleavable linker (CL); and (iii)an antibody or an antigen binding fragment thereof (AB) thatspecifically binds a target, wherein (1) the NB does not bindspecifically to the AB; (2) the CL is a polypeptide that includes asubstrate (S) for an enzyme, e.g., a protease selected from matriptaseand uPA; (3) the CL is positioned such that in an uncleaved state, theNB interferes with binding of the AB to the target and in a cleavedstate, the NB does not interfere with binding of the AB to the target;and (4) the NB does not inhibit cleavage of the CL by the enzyme; and(b) recovering the activatable antibody.

In some embodiments, the method includes the steps of (a) culturing acell that includes a nucleic acid construct that encodes the activatableantibody under conditions that lead to expression of the activatableantibody, wherein the activatable antibody includes (i) a bindingpartner (BP) for a non-binding steric moiety (NB); (ii) a cleavablelinker (CL); and (iii) an antibody or an antigen binding fragmentthereof (AB) that specifically binds a target, wherein (1) the NB doesnot bind specifically to the AB; (2) the CL is a polypeptide thatincludes a substrate (S) for an enzyme, e.g., a protease selected frommatriptase and uPA; (3) the CL is positioned such that in an uncleavedstate in the presence of the NB, the NB interferes with binding of theAB to the target and in a cleaved state, the NB does not interfere withbinding of the AB to the target and the BP does not interfere withbinding of the AB to the target; and (4) the NB and the BP do notinhibit cleavage of the CL by the enzyme; and (b) recovering theactivatable antibody. In some examples of this embodiment, the BP of theactivatable antibody is bound to the NB.

Use of CM-Containing Molecules Including Conjugated Antibodies andActivatable Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the disclosure will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present disclosure, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32 (2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203 (1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89 (8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the disclosure, which include aCM-containing molecule, such as by way of non-limiting example, aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody, are used to prevent, treat or otherwise amelioratea disease or disorder associated with aberrant target expression and/oractivity. For example, therapeutic formulations of the disclosure, whichinclude a CM-containing molecule, e.g., a conjugated antibody, anactivatable antibody and/or a conjugated activatable antibody, are usedto treat or otherwise ameliorate inflammation, an inflammatory disorder,an autoimmune disease and/or a cancer or other neoplastic condition. Insome embodiments, the cancer is a solid tumor or a hematologicmalignancy where the target is expressed. In some embodiments, thecancer is a solid tumor where the target is expressed. In someembodiments, the cancer is a hematologic malignancy where the target isexpressed. In some embodiments, the target is expressed on parenchyma(e.g., in cancer, the portion of an organ or tissue that often carriesout function(s) of the organ or tissue). In some embodiments, the targetis expressed on a cell, tissue, or organ. In some embodiments, thetarget is expressed on stroma (i.e., the connective supportive frameworkof a cell, tissue, or organ). In some embodiments, the target isexpressed on an osteoblast. In some embodiments, the target is expressedon the endothelium (vasculature). In some embodiments, the target isexpressed on a cancer stem cell. In some embodiments, the agent to whichthe activatable antibody is conjugated is a microtubule inhibitor. Insome embodiments, the agent to which the activatable antibody isconjugated is a nucleic acid damaging agent.

Efficaciousness of prevention, amelioration or treatment is determinedin association with any known method for diagnosing or treating thedisease or disorder associated with target expression and/or activity,such as, for example, aberrant target expression and/or activity.Prolonging the survival of a subject or otherwise delaying theprogression of the disease or disorder associated with target expressionand/or activity, e.g., aberrant target expression and/or activity, in asubject indicates that the conjugated antibody, activatable antibodyand/or conjugated activatable antibody confers a clinical benefit.

A CM-containing molecule, e.g., a conjugated antibody, an activatableantibody and/or a conjugated activatable antibody can be administered inthe form of pharmaceutical compositions. Principles and considerationsinvolved in preparing such compositions, as well as guidance in thechoice of components are provided, for example, in Remington: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

In some embodiments where antibody fragments are used, the smallestfragment that specifically binds to the binding domain of the targetprotein is selected. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)). The formulation can also contain more than one active compoundsas necessary for the particular indication being treated, for example,in some embodiments, those with complementary activities that do notadversely affect each other. In some embodiments, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

In some embodiments, the CM-containing molecule, e.g., the conjugatedantibody, activatable antibody and/or conjugated activatable antibodycontains a detectable label. An intact antibody, or a fragment thereof(e.g., Fab, scFv, or F(ab)₂) is used. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the disclosure can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, immunochemical staining, and immunofluorescence.In vitro techniques for detection of an analyte genomic DNA includeSouthern hybridizations. Procedures for conducting immunoassays aredescribed, for example in “ELISA: Theory and Practice: Methods inMolecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa,N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, AcademicPress, Inc., San Diego, Calif., 1996; and “Practice and Theory of EnzymeImmunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte proteininclude introducing into a subject a labeled anti-analyte proteinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

The conjugated antibodies, activatable antibodies and/or conjugatedactivatable antibodies of the disclosure are also useful in a variety ofdiagnostic and prophylactic formulations. In one embodiment, aconjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody is administered to patients that are at risk ofdeveloping one or more of the aforementioned disorders. A patient's ororgan's predisposition to one or more of the aforementioned disorderscan be determined using genotypic, serological or biochemical markers.

In some embodiments of the disclosure, a conjugated antibody, anactivatable antibody and/or a conjugated activatable antibody isadministered to human individuals diagnosed with a clinical indicationassociated with one or more of the aforementioned disorders. Upondiagnosis, a conjugated antibody, an activatable antibody and/or aconjugated activatable antibody is administered to mitigate or reversethe effects of the clinical indication.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody of the disclosure is also useful in the detectionof a target in patient samples and accordingly are useful asdiagnostics. For example, the antibodies and/or activatable antibodies,and conjugated versions thereof, of the disclosure are used in in vitroassays, e.g., ELISA, to detect target levels in a patient sample.

In one embodiment, a conjugated antibody, an activatable antibody and/ora conjugated activatable antibody of the disclosure is immobilized on asolid support (e.g., the well(s) of a microtiter plate). The immobilizedconjugated antibody, activatable antibody and/or conjugated activatableantibody serves as a capture antibody for any target that may be presentin a test sample. Prior to contacting the immobilized antibody with apatient sample, the solid support is rinsed and treated with a blockingagent such as milk protein or albumin to prevent nonspecific adsorptionof the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of target antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theantibodies of the disclosure, and conjugated versions thereof, in an invitro diagnostic assay, it is possible to stage a disease in a subjectbased on expression levels of the target antigen. For a given disease,samples of blood are taken from subjects diagnosed as being at variousstages in the progression of the disease, and/or at various points inthe therapeutic treatment of the disease. Using a population of samplesthat provides statistically significant results for each stage ofprogression or therapy, a range of concentrations of the antigen thatmay be considered characteristic of each stage is designated.

A conjugated antibody, an activatable antibody and/or a conjugatedactivatable antibody can also be used in diagnostic and/or imagingmethods. In some embodiments, such methods are in vitro methods. In someembodiments, such methods are in vivo methods. In some embodiments, suchmethods are in situ methods. In some embodiments, such methods are exvivo methods. For example, activatable antibodies having anenzymatically cleavable CM can be used to detect the presence or absenceof an enzyme that is capable of cleaving the CM. Such activatableantibodies can be used in diagnostics, which can include in vivodetection (e.g., qualitative or quantitative) of enzyme activity (or, insome embodiments, an environment of increased reduction potential suchas that which can provide for reduction of a disulfide bond) throughmeasured accumulation of activated antibodies (i.e., antibodiesresulting from cleavage of an activatable antibody) in a given cell ortissue of a given host organism. Such accumulation of activatedantibodies indicates not only that the tissue expresses enzymaticactivity (or an increased reduction potential depending on the nature ofthe CM) but also that the tissue expresses target to which the activatedantibody binds.

For example, the CM can be selected to be substrate for at least oneprotease selected from matriptase and uPA found at the site of a tumor,at the site of a viral or bacterial infection at a biologically confinedsite (e.g., such as in an abscess, in an organ, and the like), and thelike. The AB can be one that binds a target antigen. Using methods asdisclosed herein, or when appropriate, methods familiar to one skilledin the art, a detectable label (e.g., a fluorescent label or radioactivelabel or radiotracer) can be conjugated to an AB or other region of anantibody and/or activatable antibody. Suitable detectable labels arediscussed in the context of the above screening methods and additionalspecific examples are provided below. Using an AB specific to a proteinor peptide of the disease state, along with at least one proteaseselected from matriptase and uPA whose activity is elevated in thedisease tissue of interest, activatable antibodies will exhibit anincreased rate of binding to disease tissue relative to tissues wherethe CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue or is inactive (e.g., inzymogen form or in complex with an inhibitor). Since small proteins andpeptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-disease tissuesor is present in inactive conformation), accumulation of activatedantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, activatable antibodies can be used to detect thepresence or absence of a cleaving agent in a sample. For example, wherethe activatable antibodies contain a CM susceptible to cleavage by anenzyme, the activatable antibodies can be used to detect (eitherqualitatively or quantitatively) the presence of an enzyme in thesample. In another example, where the activatable antibodies contain aCM susceptible to cleavage by reducing agent, the activatable antibodiescan be used to detect (either qualitatively or quantitatively) thepresence of reducing conditions in a sample. To facilitate analysis inthese methods, the activatable antibodies can be detectably labeled, andcan be bound to a support (e.g., a solid support, such as a slide orbead). The detectable label can be positioned on a portion of theactivatable antibody that is not released following cleavage, forexample, the detectable label can be a quenched fluorescent label orother label that is not detectable until cleavage has occurred. Theassay can be conducted by, for example, contacting the immobilized,detectably labeled activatable antibodies with a sample suspected ofcontaining an enzyme and/or reducing agent for a time sufficient forcleavage to occur, then washing to remove excess sample andcontaminants. The presence or absence of the cleaving agent (e.g.,enzyme or reducing agent) in the sample is then assessed by a change indetectable signal of the activatable antibodies prior to contacting withthe sample e.g., the presence of and/or an increase in detectable signaldue to cleavage of the activatable antibody by the cleaving agent in thesample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable antibodies when cleaved. Thus, the assays can be adaptedto assess the presence or absence of a cleaving agent and the presenceor absence of a target of interest. The presence or absence of thecleaving agent can be detected by the presence of and/or an increase indetectable label of the activatable antibodies as described above, andthe presence or absence of the target can be detected by detection of atarget-AB complex e.g., by use of a detectably labeled anti-targetantibody.

Activatable antibodies are also useful in in situ imaging for thevalidation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g. afluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate(TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot®nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotinand an amplification reagent such as streptavidin, or an enzyme (e.g.horseradish peroxidase or alkaline phosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable antibody indicates that the sample contains thetarget and contains a protease selected from matriptase and uPA that isspecific for the CM of the activatable antibody. In some embodiments,the presence of the protease selected from matriptase and uPA can beconfirmed using broad spectrum protease inhibitors such as thosedescribed herein, and/or by using an agent that is specific for theprotease, for example, an antibody such as A11, which is specific forthe protease matriptase and inhibits the proteolytic activity ofmatriptase; see e.g., International Publication Number WO 2010/129609,published 11 Nov. 2010. The same approach of using broad spectrumprotease inhibitors such as those described herein, and/or by using amore selective inhibitory agent can be used to identify a proteaseselected from matriptase and uPA that is specific for the CM of theactivatable antibody. In some embodiments, the presence of the targetcan be confirmed using an agent that is specific for the target, e.g.,another antibody, or the detectable label can be competed with unlabeledtarget. In some embodiments, unlabeled activatable antibody could beused, with detection by a labeled secondary antibody or more complexdetection system.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target and containsa protease selected from matriptase and uPA that is specific for the CMof the activatable antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable antibody.

The disclosure provides methods of using the antibodies and/oractivatable antibodies in a variety of diagnostic and/or prophylacticindications. For example, the disclosure provides methods of detectingpresence or absence of a cleaving agent and a target of interest in asubject or a sample by (i) contacting a subject or sample with anactivatable antibody, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, e.g., a protease selected from matriptase and uPA, andan antigen binding domain or fragment thereof (AB) that specificallybinds the target of interest, wherein the activatable antibody in anuncleaved, non-activated state comprises a structural arrangement fromN-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) whereinthe MM1 is a peptide that inhibits binding of the AB to the target, andwherein the MM1 does not have an amino acid sequence of a naturallyoccurring binding partner of the AB and is not a modified form of anatural binding partner of the AB; and (b) wherein, in an uncleaved,non-activated state, the MM interferes with specific binding of the ABto the target, and in a cleaved, activated state the MM1 does notinterfere or compete with specific binding of the AB to the target; and(ii) measuring a level of activated activatable antibody in the subjector sample, wherein a detectable level of activated activatable antibodyin the subject or sample indicates that the cleaving agent and thetarget are present in the subject or sample and wherein no detectablelevel of activated activatable antibody in the subject or sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector sample. In some embodiments, the activatable antibody is anactivatable antibody to which a therapeutic agent is conjugated. In someembodiments, the activatable antibody is not conjugated to an agent. Insome embodiments, the activatable antibody comprises a detectable label.In some embodiments, the detectable label is positioned on the AB. Insome embodiments, measuring the level of activatable antibody in thesubject or sample is accomplished using a secondary reagent thatspecifically binds to the activated antibody, wherein the reagentcomprises a detectable label. In some embodiments, the secondary reagentis an antibody comprising a detectable label.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable antibody in the presence of a target ofinterest, e.g., the target, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, e.g., a protease selected from matriptase and uPA, andan antigen binding domain or fragment thereof (AB) that specificallybinds the target of interest, wherein the activatable antibody in anuncleaved, non-activated state comprises a structural arrangement fromN-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) whereinthe MM1 is a peptide that inhibits binding of the AB to the target, andwherein the MM1 does not have an amino acid sequence of a naturallyoccurring binding partner of the AB and is not a modified form of anatural binding partner of the AB; and (b) wherein, in an uncleaved,non-activated state, the MM interferes with specific binding of the ABto the target, and in a cleaved, activated state the MM1 does notinterfere or compete with specific binding of the AB to the target; and(ii) measuring a level of activated activatable antibody in the subjector sample, wherein a detectable level of activated activatable antibodyin the subject or sample indicates that the cleaving agent is present inthe subject or sample and wherein no detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent is absent and/or not sufficiently present in the subjector sample. In some embodiments, the activatable antibody is anactivatable antibody to which a therapeutic agent is conjugated. In someembodiments, the activatable antibody is not conjugated to an agent. Insome embodiments, the activatable antibody comprises a detectable label.In some embodiments, the detectable label is positioned on the AB. Insome embodiments, measuring the level of activatable antibody in thesubject or sample is accomplished using a secondary reagent thatspecifically binds to the activated antibody, wherein the reagentcomprises a detectable label. In some embodiments, the secondary reagentis an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, e.g., a protease selected from matriptase anduPA, and an antigen binding domain or fragment thereof (AB) thatspecifically binds the target of interest, wherein the activatableantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM1 is a peptide that inhibits binding of theAB to the target, and wherein the MM1 does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the target, and in a cleaved, activated state theMM1 does not interfere or compete with specific binding of the AB to thetarget; and (ii) measuring a level of activated activatable antibody inthe subject or sample, wherein a detectable level of activatedactivatable antibody in the subject or sample indicates that thecleaving agent is present in the subject or sample and wherein nodetectable level of activated activatable antibody in the subject orsample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or sample. In some embodiments, theactivatable antibody is an activatable antibody to which a therapeuticagent is conjugated. In some embodiments, the activatable antibody isnot conjugated to an agent. In some embodiments, the activatableantibody comprises a detectable label. In some embodiments, thedetectable label is positioned on the AB. In some embodiments, measuringthe level of activatable antibody in the subject or sample isaccomplished using a secondary reagent that specifically binds to theactivated antibody, wherein the reagent comprises a detectable label. Insome embodiments, the secondary reagent is an antibody comprising adetectable label.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable antibody, wherein the activatable antibodycomprises a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, e.g., a protease selected from matriptase anduPA, an antigen binding domain (AB) that specifically binds the target,and a detectable label, wherein the activatable antibody in anuncleaved, non-activated state comprises a structural arrangement fromN-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; wherein theMM is a peptide that inhibits binding of the AB to the target, andwherein the MM does not have an amino acid sequence of a naturallyoccurring binding partner of the AB and is not a modified form of anatural binding partner of the AB; wherein, in an uncleaved,non-activated state, the MM interferes with specific binding of the ABto the target, and in a cleaved, activated state the MM does notinterfere or compete with specific binding of the AB to the target; andwherein the detectable label is positioned on a portion of theactivatable antibody that is released following cleavage of the CM; and(ii) measuring a level of detectable label in the subject or sample,wherein a detectable level of the detectable label in the subject orsample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or sample and wherein no detectablelevel of the detectable label in the subject or sample indicates thatthe cleaving agent is present in the subject or sample. In someembodiments, the activatable antibody is an activatable antibody towhich a therapeutic agent is conjugated. In some embodiments, theactivatable antibody is not conjugated to an agent. In some embodiments,the activatable antibody comprises a detectable label. In someembodiments, the detectable label is positioned on the AB. In someembodiments, measuring the level of activatable antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody (e.g., an activatable antibody to whicha therapeutic agent is conjugated) described herein for use incontacting a subject or biological sample and means for detecting thelevel of activated activatable antibody and/or conjugated activatableantibody in the subject or biological sample, wherein a detectable levelof activated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample and wherein no detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable antibody in the presence of thetarget, and (ii) measuring a level of activated activatable antibody inthe subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent is present in the subject orbiological sample and wherein no detectable level of activatedactivatable antibody in the subject or biological sample indicates thatthe cleaving agent is absent and/or not sufficiently present in thesubject or biological sample at a detectable level, such that proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample. Such an activatable antibody includes a maskingmoiety (MM), a cleavable moiety (CM) that is cleaved by the cleavingagent, e.g., a protease selected from matriptase and uPA, and an antigenbinding domain or fragment thereof (AB) that specifically binds thetarget, wherein the activatable antibody in an uncleaved (i.e.,non-activated) state comprises a structural arrangement from N-terminusto C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is apeptide that inhibits binding of the AB to the target, and wherein theMM does not have an amino acid sequence of a naturally occurring bindingpartner of the AB; and (b) wherein the MM of the activatable antibody inan uncleaved state interferes with specific binding of the AB to thetarget, and wherein the MM of an activatable antibody in a cleaved(i.e., activated) state does not interfere or compete with specificbinding of the AB to the target. In some embodiments, the activatableantibody is an activatable antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable antibody is notconjugated to an agent. In some embodiments, the detectable label isattached to the masking moiety. In some embodiments, the detectablelabel is attached to the cleavable moiety N-terminal to the proteasecleavage site. In some embodiments, a single antigen binding site of theAB is masked. In some embodiments wherein an antibody of the disclosurehas at least two antigen binding sites, at least one antigen bindingsite is masked and at least one antigen binding site is not masked. Insome embodiments all antigen binding sites are masked. In someembodiments, the measuring step includes use of a secondary reagentcomprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample with an activatable antibody in thepresence of the target, and measuring a level of activated activatableantibody in the subject or biological sample, wherein a detectable levelof activated activatable antibody in the subject or biological sampleindicates that the cleaving agent is present in the subject orbiological sample and wherein no detectable level of activatedactivatable antibody in the subject or biological sample indicates thatthe cleaving agent is absent and/or not sufficiently present in thesubject or biological sample at a detectable level, such that proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample. Such an activatable antibody includes a maskingmoiety (MM), a cleavable moiety (CM) that is cleaved by the cleavingagent, e.g., a protease selected from matriptase and uPA, and an antigenbinding domain or fragment thereof (AB) that specifically binds thetarget, wherein the activatable antibody in an uncleaved (i.e.,non-activated) state comprises a structural arrangement from N-terminusto C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM1 is apeptide that inhibits binding of the AB to the target, and wherein theMM1 does not have an amino acid sequence of a naturally occurringbinding partner of the AB; and (b) wherein the MM1 of the activatableantibody in an uncleaved state interferes with specific binding of theAB to the target, and wherein the MM of an activatable antibody in acleaved (i.e., activated) state does not interfere or compete withspecific binding of the AB to the target. In some embodiments, theactivatable antibody is an activatable antibody to which a therapeuticagent is conjugated. In some embodiments, the activatable antibody isnot conjugated to an agent. In some embodiments, the detectable label isattached to the masking moiety. In some embodiments, the detectablelabel is attached to the cleavable moiety N-terminal to the proteasecleavage site. In some embodiments, a single antigen binding site of theAB is masked. In some embodiments wherein an antibody of the disclosurehas at least two antigen binding sites, at least one antigen bindingsite is masked and at least one antigen binding site is not masked. Insome embodiments all antigen binding sites are masked. In someembodiments, the measuring step includes use of a secondary reagentcomprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent in a subject or a sample, wherethe kits include at least an activatable antibody and/or conjugatedactivatable antibody described herein for use in contacting a subject orbiological sample and means for detecting the level of activatedactivatable antibody and/or conjugated activatable antibody in thesubject or biological sample, wherein the activatable antibody includesa detectable label that is positioned on a portion of the activatableantibody that is released following cleavage of the CM, wherein adetectable level of activated activatable antibody in the subject orbiological sample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or biological sample such that thetarget binding and/or protease cleavage of the activatable antibodycannot be detected in the subject or biological sample, and wherein nodetectable level of activated activatable antibody in the subject orbiological sample indicates that the cleaving agent is present in thesubject or biological sample at a detectable level.

The disclosure provides methods of detecting presence or absence of acleaving agent and the target in a subject or a sample by (i) contactinga subject or biological sample with an activatable antibody, wherein theactivatable antibody includes a detectable label that is positioned on aportion of the activatable antibody that is released following cleavageof the CM and (ii) measuring a level of activated activatable antibodyin the subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample, and wherein a reduced detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample. A reduced level of detectable label is,for example, a reduction of about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95% and/or about 100%. Such an activatable antibodyincludes a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, and an antigen binding domain or fragment thereof(AB) that specifically binds the target, wherein the activatableantibody in an uncleaved (i.e., non-activated) state comprises astructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibitsbinding of the AB to the target, and wherein the MM does not have anamino acid sequence of a naturally occurring binding partner of the AB;and (b) wherein the MM of the activatable antibody in an uncleaved stateinterferes with specific binding of the AB to the target, and whereinthe MM of an activatable antibody in a cleaved (i.e., activated) statedoes not interfere or compete with specific binding of the AB to thetarget. In some embodiments, the activatable antibody is an activatableantibody to which a therapeutic agent is conjugated. In someembodiments, the activatable antibody is not conjugated to an agent. Insome embodiments, the activatable antibody comprises a detectable label.In some embodiments, the detectable label is positioned on the AB. Insome embodiments, measuring the level of activatable antibody in thesubject or sample is accomplished using a secondary reagent thatspecifically binds to the activated antibody, wherein the reagentcomprises a detectable label. In some embodiments, the secondary reagentis an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent and the target in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample and means for detecting the level ofactivated activatable antibody and/or conjugated activatable antibody inthe subject or biological sample, wherein a detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent, the target or both the cleaving agentand the target are absent and/or not sufficiently present in the subjector biological sample, such that the target binding and/or proteasecleavage of the activatable antibody cannot be detected in the subjector biological sample, and wherein a reduced detectable level ofactivated activatable antibody in the subject or biological sampleindicates that the cleaving agent and the target are present in thesubject or biological sample. A reduced level of detectable label is,for example, a reduction of about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95% and/or about 100%.

The disclosure also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable antibody, wherein the activatableantibody includes a detectable label that is positioned on a portion ofthe activatable antibody that is released following cleavage of the CM;and (ii) measuring a level of detectable label in the subject orbiological sample, wherein a detectable level of the detectable label inthe subject or biological sample indicates that the cleaving agent isabsent and/or not sufficiently present in the subject or biologicalsample at a detectable level, such that protease cleavage of theactivatable antibody cannot be detected in the subject or biologicalsample, and wherein a reduced detectable level of the detectable labelin the subject or biological sample indicates that the cleaving agent ispresent in the subject or biological sample. A reduced level ofdetectable label is, for example, a reduction of about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95% and/or about 100%. Such anactivatable antibody includes a masking moiety (MM), a cleavable moiety(CM) that is cleaved by the cleaving agent, and an antigen bindingdomain or fragment thereof (AB) that specifically binds the target,wherein the activatable antibody in an uncleaved (i.e., non-activated)state comprises a structural arrangement from N-terminus to C-terminusas follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide thatinhibits binding of the AB to the target, and wherein the MM does nothave an amino acid sequence of a naturally occurring binding partner ofthe AB; and (b) wherein the MM of the activatable antibody in anuncleaved state interferes with specific binding of the AB to thetarget, and wherein the MM of an activatable antibody in a cleaved(i.e., activated) state does not interfere or compete with specificbinding of the AB to the target. In some embodiments, the activatableantibody is an activatable antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable antibody is notconjugated to an agent. In some embodiments, the activatable antibodycomprises a detectable label. In some embodiments, the detectable labelis positioned on the AB. In some embodiments, measuring the level ofactivatable antibody in the subject or sample is accomplished using asecondary reagent that specifically binds to the activated antibody,wherein the reagent comprises a detectable label. In some embodiments,the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detectingpresence or absence of a cleaving agent of interest in a subject or asample, where the kits include at least an activatable antibody and/orconjugated activatable antibody described herein for use in contacting asubject or biological sample and means for detecting the level ofactivated activatable antibody and/or conjugated activatable antibody inthe subject or biological sample, wherein the activatable antibodyincludes a detectable label that is positioned on a portion of theactivatable antibody that is released following cleavage of the CM,wherein a detectable level of the detectable label in the subject orbiological sample indicates that the cleaving agent, the target, or boththe cleaving agent and the target are absent and/or not sufficientlypresent in the subject or biological sample, such that the targetbinding and/or protease cleavage of the activatable antibody cannot bedetected in the subject or biological sample, and wherein a reduceddetectable level of the detectable label in the subject or biologicalsample indicates that the cleaving agent and the target are present inthe subject or biological sample. A reduced level of detectable labelis, for example, a reduction of about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95% and/or about 100%.

In some embodiments of these methods and kits, the activatable antibodyincludes a detectable label. In some embodiments of these methods andkits, the detectable label includes an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, or a ligand-based label. In some embodiments of thesemethods and kits, the imaging agent comprises a radioisotope. In someembodiments of these methods and kits, the radioisotope is indium ortechnetium. In some embodiments of these methods and kits, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods and kits, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or β-galactosidase. In someembodiments of these methods and kits, the fluorescent label comprisesyellow fluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods and kits, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods and kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods and kits, the subject is a human.In some embodiments, the subject is a non-human mammal, such as anon-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. In some embodiments, the subject isa rodent.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable antibodies are used to screen patient samplesto identify those patients having the appropriate protease(s) andtarget(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target (e.g., the target) and a protease that cleaves thesubstrate in the cleavable moiety (CM) of the activatable antibody beingtested (e.g., accumulate activated antibodies at the disease site) areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM. Likewise, patients that test negative foreither or both of the target (e.g., the target) and the protease thatcleaves the substrate in the CM in the activatable antibody being testedusing these methods might be identified as suitable candidates foranother form of therapy. In some embodiments, such patients that testnegative with respect to a first activatable antibody can be tested withother activatable antibodies comprising different CMs until a suitableactivatable antibody for treatment is identified (e.g., an activatableantibody comprising a CM that is cleaved by the patient at the site ofdisease). In some embodiments, the patient is then administered atherapeutically effective amount of the conjugated activatable antibodyfor which the patient tested positive.

In some embodiments in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target (e.g., the target) and a protease that cleaves thesubstrate in the cleavable moiety (CM) of the activatable antibody beingtested (e.g., accumulate activated antibodies at the disease site) areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM. Likewise, patients that test negativemight be identified as suitable candidates for another form of therapy.In some embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).In some embodiments, the patient is then administered a therapeuticallyeffective amount of the conjugated activatable antibody for which thepatient tested positive.

In some embodiments of the methods and kits, the method or kit is usedto identify or otherwise refine a patient population suitable fortreatment with an activatable antibody of the disclosure. For example,patients that test positive for both the target (e.g., the target) and aprotease that cleaves the substrate in the cleavable moiety (CM) of theactivatable antibody being tested in these methods are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative for both ofthe targets (e.g., the target) and the protease that cleaves thesubstrate in the CM in the activatable antibody being tested using thesemethods might be identified as suitable candidates for another form oftherapy. In some embodiments, such patients can be tested with otheractivatable antibodies until a suitable activatable antibody fortreatment is identified (e.g., an activatable antibody comprising a CMthat is cleaved by the patient at the site of disease). In someembodiments, patients that test negative for either of the target (e.g.,the target) are identified as suitable candidates for treatment withsuch an activatable antibody comprising such a CM. In some embodiments,patients that test negative for either of the target (e.g., the target)are identified as not being suitable candidates for treatment with suchan activatable antibody comprising such a CM. In some embodiments, suchpatients can be tested with other activatable antibodies until asuitable activatable antibody for treatment is identified (e.g., anactivatable antibody comprising a CM that is cleaved by the patient atthe site of disease). In some embodiments, the activatable antibody isan activatable antibody to which a therapeutic agent is conjugated. Insome embodiments, the activatable antibody is not conjugated to anagent. In some embodiments, the activatable antibody comprises adetectable label. In some embodiments, the detectable label ispositioned on the AB. In some embodiments, measuring the level ofactivatable antibody in the subject or sample is accomplished using asecondary reagent that specifically binds to the activated antibody,wherein the reagent comprises a detectable label. In some embodiments,the secondary reagent is an antibody comprising a detectable label.

In some embodiments, a method or kit is used to identify or otherwiserefine a patient population suitable for treatment with an anti—thetarget activatable antibody and/or conjugated activatable antibody(e.g., activatable antibody to which a therapeutic agent is conjugated)of the disclosure, followed by treatment by administering thatactivatable antibody and/or conjugated activatable antibody to a subjectin need thereof. For example, patients that test positive for both thetargets (e.g., the target) and a protease that cleaves the substrate inthe cleavable moiety (CM) of the activatable antibody and/or conjugatedactivatable antibody being tested in these methods are identified assuitable candidates for treatment with such antibody and/or such aconjugated activatable antibody comprising such a CM, and the patient isthen administered a therapeutically effective amount of the activatableantibody and/or conjugated activatable antibody that was tested.Likewise, patients that test negative for either or both of the target(e.g., the target) and the protease that cleaves the substrate in the CMin the activatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other antibody and/orconjugated activatable antibody until a suitable antibody and/orconjugated activatable antibody for treatment is identified (e.g., anactivatable antibody and/or conjugated activatable antibody comprising aCM that is cleaved by the patient at the site of disease). In someembodiments, the patient is then administered a therapeuticallyeffective amount of the activatable antibody and/or conjugated for whichthe patient tested positive.

In some embodiments of these methods and kits, the MM is a peptidehaving a length from about 4 to 40 amino acids. In some embodiments ofthese methods and kits, the activatable antibody comprises a linkerpeptide, wherein the linker peptide is positioned between the MM and theCM. In some embodiments of these methods and kits, the activatableantibody comprises a linker peptide, where the linker peptide ispositioned between the AB and the CM. In some embodiments of thesemethods and kits, the activatable antibody comprises a first linkerpeptide (L1) and a second linker peptide (L2), wherein the first linkerpeptide is positioned between the MM and the CM and the second linkerpeptide is positioned between the AB and the CM. In some embodiments ofthese methods and kits, each of L1 and L2 is a peptide of about 1 to 20amino acids in length, and wherein each of L1 and L2 need not be thesame linker. In some embodiments of these methods and kits, one or bothof L1 and L2 comprises a glycine-serine polymer. In some embodiments ofthese methods and kits, at least one of L1 and L2 comprises an aminoacid sequence selected from the group consisting of (GS)n, (GSGGS)n (SEQID NO: 385) and (GGGS)n (SEQ ID NO: 386), where n is an integer of atleast one. In some embodiments of these methods and kits, at least oneof L1 and L2 comprises an amino acid sequence having the formula (GGS)n,where n is an integer of at least one. In some embodiments of thesemethods and kits, at least one of L1 and L2 comprises an amino acidsequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ IDNO: 387), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 388), Gly-Ser-Gly-Ser-Gly (SEQID NO: 389), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 390), Gly-Gly-Gly-Ser-Gly(SEQ ID NO: 391), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 392).

In some embodiments of these methods and kits, the AB comprises anantibody or antibody fragment sequence selected from the cross-reactiveantibody sequences presented herein. In some embodiments of thesemethods and kits, the AB comprises a Fab fragment, a scFv or a singlechain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is aprotease that is co-localized in the subject or sample with the targetand the CM is a polypeptide that functions as a substrate for theprotease, wherein the protease cleaves the CM in the activatableantibody when the activatable antibody is exposed to the protease. Insome embodiments of these methods and kits, the CM is a polypeptide ofup to 15 amino acids in length. In some embodiments of these methods andkits, the CM is coupled to the N-terminus of the AB. In some embodimentsof these methods and kits, the CM is coupled to the C-terminus of theAB. In some embodiments of these methods and kits, the CM is coupled tothe N-terminus of a VL chain of the AB.

The activatable antibodies and/or conjugated activatable antibodies ofthe disclosure are used in diagnostic and prophylactic formulations. Inone embodiment, an activatable antibody is administered to patients thatare at risk of developing one or more of the aforementionedinflammation, inflammatory disorders, cancer or other disorders.

A patient's or organ's predisposition to one or more of theaforementioned disorders can be determined using genotypic, serologicalor biochemical markers.

In some embodiments of the disclosure, an activatable antibody and/orconjugated activatable antibodies is administered to human individualsdiagnosed with a clinical indication associated with one or more of theaforementioned disorders. Upon diagnosis, an activatable antibody and/orconjugated activatable antibodies is administered to mitigate or reversethe effects of the clinical indication.

Activatable antibodies and/or conjugated activatable antibodies of thedisclosure are also useful in the detection of the target in patientsamples and accordingly are useful as diagnostics. For example, theactivatable antibodies and/or conjugated activatable antibodies of thedisclosure are used in in vitro assays, e.g., ELISA, to detect targetlevels in a patient sample.

In one embodiment, an activatable antibody of the disclosure isimmobilized on a solid support (e.g., the well(s) of a microtiterplate). The immobilized activatable antibody serves as a captureantibody for any target that may be present in a test sample. Prior tocontacting the immobilized antibody with a patient sample, the solidsupport is rinsed and treated with a blocking agent such as milk proteinor albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of target antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theantibodies of the disclosure in an in vitro diagnostic assay, it ispossible to stage a disease in a subject based on expression levels ofthe Target antigen. For a given disease, samples of blood are taken fromsubjects diagnosed as being at various stages in the progression of thedisease, and/or at various points in the therapeutic treatment of thedisease. Using a population of samples that provides statisticallysignificant results for each stage of progression or therapy, a range ofconcentrations of the antigen that may be considered characteristic ofeach stage is designated.

Activatable antibodies and/or conjugated activatable antibodies can alsobe used in diagnostic and/or imaging methods. In some embodiments, suchmethods are in vitro methods. In some embodiments, such methods are invivo methods. In some embodiments, such methods are in situ methods. Insome embodiments, such methods are ex vivo methods. For example,activatable antibodies having an enzymatically cleavable CM can be usedto detect the presence or absence of an enzyme that is capable ofcleaving the CM. Such activatable antibodies can be used in diagnostics,which can include in vivo detection (e.g., qualitative or quantitative)of enzyme activity (or, in some embodiments, an environment of increasedreduction potential such as that which can provide for reduction of adisulfide bond) through measured accumulation of activated antibodies(i.e., antibodies resulting from cleavage of an activatable antibody) ina given cell or tissue of a given host organism. Such accumulation ofactivated antibodies indicates not only that the tissue expressesenzymatic activity (or an increased reduction potential depending on thenature of the CM) but also that the tissue expresses target to which theactivated antibody binds.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label or radioactivelabel or radiotracer) can be conjugated to an AB or other region of anactivatable antibody. Suitable detectable labels are discussed in thecontext of the above screening methods and additional specific examplesare provided below. Using an AB specific to a protein or peptide of thedisease state, along with a protease whose activity is elevated in thedisease tissue of interest, activatable antibodies will exhibit anincreased rate of binding to disease tissue relative to tissues wherethe CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue or is inactive (e.g., inzymogen form or in complex with an inhibitor). Since small proteins andpeptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-disease tissuesor is present in inactive conformation), accumulation of activatedantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, activatable antibodies can be used to detect thepresence or absence of a cleaving agent in a sample. For example, wherethe activatable antibodies contain a CM susceptible to cleavage by anenzyme, the activatable antibodies can be used to detect (eitherqualitatively or quantitatively) the presence of an enzyme in thesample. In another example, where the activatable antibodies contain aCM susceptible to cleavage by reducing agent, the activatable antibodiescan be used to detect (either qualitatively or quantitatively) thepresence of reducing conditions in a sample. To facilitate analysis inthese methods, the activatable antibodies can be detectably labeled, andcan be bound to a support (e.g., a solid support, such as a slide orbead). The detectable label can be positioned on a portion of theactivatable antibody that is not released following cleavage, forexample, the detectable label can be a quenched fluorescent label orother label that is not detectable until cleavage has occurred. Theassay can be conducted by, for example, contacting the immobilized,detectably labeled activatable antibodies with a sample suspected ofcontaining an enzyme and/or reducing agent for a time sufficient forcleavage to occur, then washing to remove excess sample andcontaminants. The presence or absence of the cleaving agent (e.g.,enzyme or reducing agent) in the sample is then assessed by a change indetectable signal of the activatable antibodies prior to contacting withthe sample e.g., the presence of and/or an increase in detectable signaldue to cleavage of the activatable antibody by the cleaving agent in thesample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable antibodies when cleaved. Thus, the assays can be adaptedto assess the presence or absence of a cleaving agent and the presenceor absence of a target of interest. The presence or absence of thecleaving agent can be detected by the presence of and/or an increase indetectable label of the activatable antibodies as described above, andthe presence or absence of the target can be detected by detection of atarget-AB complex e.g., by use of a detectably labeled anti-targetantibody.

Activatable antibodies are also useful in in situ imaging for thevalidation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g.Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), anear infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, ahapten, a radioactive marker, biotin and an amplification reagent suchas streptavidin, or an enzyme (e.g. horseradish peroxidase or alkalinephosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable antibody indicates that the sample contains thetarget and contains a protease that is specific for the CM of theactivatable antibody. In some embodiments, the presence of the proteasecan be confirmed using broad spectrum protease inhibitors such as thosedescribed herein, and/or by using an agent that is specific for theprotease, for example, an antibody such as A11, which is specific forthe protease matriptase and inhibits the proteolytic activity ofmatriptase; see e.g., International Publication Number WO 2010/129609,published 11 Nov. 2010. The same approach of using broad spectrumprotease inhibitors such as those described herein, and/or by using amore selective inhibitory agent can be used to identify a protease orclass of proteases specific for the CM of the activatable antibody. Insome embodiments, the presence of the target can be confirmed using anagent that is specific for the target, e.g., another antibody, or thedetectable label can be competed with unlabeled target. In someembodiments, unlabeled activatable antibody could be used, withdetection by a labeled secondary antibody or more complex detectionsystem.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target and containsa protease that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable antibody.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable antibodies are used to screen patient samplesto identify those patients having the appropriate protease(s) andtarget(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target and a protease that cleaves the substrate in thecleavable moiety (CM) of the activatable antibody being tested (e.g.,accumulate activated antibodies at the disease site) are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative for eitheror both of the target and the protease that cleaves the substrate in theCM in the activatable antibody being tested using these methods areidentified as suitable candidates for another form of therapy (i.e., notsuitable for treatment with the activatable antibody being tested). Insome embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

In some embodiments in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target and a protease that cleaves the substrate in thecleavable moiety (CM) of the activatable antibody being tested (e.g.,accumulate activated antibodies at the disease site) are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative areidentified as suitable candidates for another form of therapy (i.e., notsuitable for treatment with the activatable antibody being tested). Insome embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

Pharmaceutical Compositions

The conjugated antibodies, activatable antibodies and/or conjugatedactivatable antibodies of the disclosure (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the conjugatedantibody, activatable antibody and/or conjugated activatable antibodyand a pharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Suitable examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In someembodiments, it will be desirable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1. Materials and Methods

Reagents and Strains:

Streptavidin-conjugated phycoerythrin (SA-PE) (Invitrogen, LifeTechnologies) was used without modifications. Human matriptase-1(Research & Diagnostics Systems, Inc.) was used without modifications.Human plasmin (Haematologic Technologies Inc.) was used withoutmodifications. Human tPA (Molecular Innovations) was used withoutmodifications. YPet fused to the SH3 domain of Mona (monocytic adaptor)was used without modifications. TBST, 50 mM Tris-HCl, 150 mM NaCl, 0.05%Tween20, pH 7.4, was used. E. coli MC1061 (Casadaban et al., JMB 138(2):179-207 (1980) was used. All bacterial growth was performed at 37°C. with vigorous shaking in Luria-Bertani broth (LB) supplemented with34 μg/mL chloramphenicol, unless another antibiotic is specified.

Substrate Cleavage and Scaffold Stability Analysis:

For screening and clone analysis, overnight cultures were subcultured bydilution into fresh medium (1:50) and grown for 1.5-2 hours. Eachsubculture was then induced with 0.04% arabinose and incubated withshaking at 37° C. for 1 hour. To stop further growth, cells wereincubated on ice for 15-30 minutes. Cell aliquots were harvested andwashed with PBS (pH 7.4). Cells were pelleted by centrifugation, thesupernatant removed, and the cells resuspended in reaction buffercontaining the enzyme; the reaction mixture was incubated at 37° C.static. To stop the reaction cells, were removed and diluted 10-fold inPBS, pelleted by centrifugation, and resuspended in PBS containingeither SA-PE (20 μg/mL) or YPet-MONA (50 nM). After incubation on ice(30 min), cells were washed with PBS and analyzed using a FACSAria™ cellsorter.

For protease cleavage assays, cultures were induced for 1 hour. Thereaction buffer for matriptase-1 was TBST. Assays for matriptase-1hydrolysis, were performed after reactions with 200 pM-200 nMmatriptase-1 for 1 hr. Background hydrolysis of the regions flanking thesubstrate site (using platform eCLiPS3.0-NSUB_SP described in PCT patentapplication PCT/US13/54378, filed Aug. 9, 2013 and published as WO2014/026136 on Feb. 13, 2014), was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion.

For human plasmin stability assays, platform eCLiPS3.0-NSUB_SP was used;cultures were induced for 1 hr. The reaction buffer for plasmin was 50mM Tris-HCl pH 7.5 supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mMEDTA. Assays for plasmin hydrolysis were performed after reactions withplasmin for 1 hr.

For human tPA stability assays, platform eCLiPS3.0-NSUB_SP was used;cultures were induced for 1 hr. The reaction buffer for tPA was TBST.Assays for tPA hydrolysis were performed after reactions with tPA for 1hr.

Amino and Carboxy Terminus Labeling Conditions:

Streptavidin conjugated phycoerythrin (SAPE) was used for labelingstreptavidin binding affinity ligand on the N-termini of CPX.Fluorescent protein YPet fused to the SH3 domain of Mona was used forlabeling the MONA binding affinity ligand on the C-termini of CPX. Foroptimum labeling of cells without protease reaction, the cells wereincubated for 30 min at 4° C. with SAPE (20 μg/mL) or YPet-MONA (50 nM).

Kinetic Data Analysis:

The extent of conversion of cell surface displayed peptide substrateswas measured directly, using flow cytometry to measure changes in meanfluorescence of clonal cell populations upon protease treatment.Specifically, for each sample, conversion was determined by flowcytometry analyses using the relationship

$\begin{matrix}{{Conversion} = \frac{{FL}_{-} - {FL}_{+}}{{FL}_{-} - {FL}_{0}}} & \lbrack 1\rbrack\end{matrix}$

where (FL⁻) is the fluorescence after incubating without enzyme, (FL₊)is fluorescence after incubation with enzyme, and (FL₀) is fluorescenceof unlabeled cells. Given that the expected substrate concentrationsthat were used are significantly below the expected K_(M) of thesubstrate for the target protease, the Michaelis-Menton model simplifiesto:

$\begin{matrix}{\frac{d\lbrack S\rbrack}{d\; t} \approx {- {{\frac{k_{cat}}{k_{M}}\lbrack S\rbrack}\lbrack E\rbrack}}} & \lbrack 2\rbrack\end{matrix}$

allowing substrate conversion to be expressed as

$\begin{matrix}{{Conversion} = {1 - {\exp \left( {{- \frac{k_{cat}}{k_{M}}} \cdot \lbrack E\rbrack \cdot t} \right)}}} & \lbrack 3\rbrack\end{matrix}$

where [S] is the substrate concentration, [E] is enzyme concentrationand t is time. To determine the second order rate constant(k_(cat)/K_(M)), equation [3] was simplified to:

$\frac{kcat}{k\; m} = {{- {\ln \left( {1 - {{product}\mspace{14mu} {conversion}}} \right)}}/\left( {{time}*\lbrack{protease}\rbrack} \right)}$

Sequence Data Analysis—Meta Motifs:

Substrates were submitted to Ion Torrent™ sequencing (see, e.g.,Rothenberg, J M, Nature 475, 348-352). Raw Ion Torrent reads werecropped by invariant vector sequences to obtain just the variablepeptide insert. Insert sequences were translated, and sequences withstop codons were excluded from further analysis. The frequency of eachsequence was obtained by number of times observed out of all viablepeptide reads observed. Enrichment of sequences was obtained bycomparison of observed frequency of each sequence post selection to thefrequency of each sequence pre-selection. Motif analysis was performedby extracting all possible 2mers, 3mers, 4mers and non-consecutive1n2mers 2n1mers 2n2mers and 2nn2mers (where the first number representsthe first set of invariant positions, the second number represents thesecond set of invariant positions, and the number of n's betweenrepresents the number of variable positions allowed between the twoinvariant positions). The frequency of each motif was established acrossall normalized sequences in both the pre-selected and post selectedlibraries to establish significance of enrichment of each motif. Togenerate meta-motifs, all sequences containing each motif were aligned,and a Positional Weight Matrix (PWM) was created representing the aminoacid propensity at each position in the motif carriers. Profile-profilealignments and scoring were conducted across all motifs, using a MinimumMutual Information Content (MMIC) scoring function to score eachprofile-profile aligned register. Registers aligning above a backgroundof incorrect registers of unique formed PWMs were extracted assignificant; the individual PWMs were then added to create an averagemetamotif.

Sequence Data Analysis—Directed Families:

Final substrate pools were sequenced using Ion Torrent™ sequencing.Individual sequences were identified and isolated from these data, andsequences were aligned in CLC main lab (CLC Main Workbench 6.6.2,available online). The alignment file was imported to Jalview (see,e.g., Waterhouse, A. M., et al., 2009, Bioinformatics 9, 1189-1191), andan average distance tree was assembled using the BLOSUM62 algorithm (SHenikoff S et al., 1992, Proc Natl Acad Sci USA. 89, 10915-10919). Therestricted group of sequences includes members of the cluster closest tothe sequence of interest. The extended group of sequences includes therestricted group of sequences those plus of the branch that shares theclosest common ancestor (where applicable).

Example 2. Selection and Characterization of Substrate Pools in aPlatform Scaffold

The use of multi-copy substrate display on whole cells enabled selectionof populations of substrates cleaved by matriptase-1. Selections wereperformed as described in U.S. Pat. No. 7,666,817 B, issued Feb. 23,2010, using recombinant human matriptase-1. Background hydrolysis of theregions flanking the substrate site (using platform eCLiPS3.0-NSUB_SP)was measured under each reaction condition to ensure that hydrolysisoccurred in the designated substrate region. Final pools were testedagainst matriptase-1, plasmin and tPA. The pools were cleaved bymatriptase-1 but not by tPA or plasmin. FIG. 1 shows cleavage of poolSMP30 by matriptase-1 in TBST, FIG. 2 shows cleavage of pool SMP17 bymatriptase-1 at 50 nM and resistance to tPA, both in TBST. Using similartechniques, a separate library was screened to select substrates cleavedby both matriptase-1 and u-plasminogen activator but not by tPA orplasmin.

Example 3. Characterization of Substrate Cleavage Kinetics in thePlatform Scaffold

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics.Consequently, flow cytometry was used to rank individual isolated cloneson the basis of substrate conversion, and clones were identified by DNAsequencing. In this way, the extent of conversion for each clone couldbe determined at several different protease concentrations and fit to aMichaelis-Menton model (Kinetic Data Analysis Section). The observedsecond order rate constant (k_(cat)/K_(M)) was determined for eachsubstrate versus matriptase-1. Background hydrolysis of the regionsflanking the substrate site (using platform eCLiPS3.0-NSUB_SP), wasmeasured under each reaction condition to ensure that hydrolysisoccurred in the designated substrate region. For example, FIG. 3 showscleavage of a substrate comprising amino acid sequence VAGRSMRP bymatriptase-1 in TBST.

Example 4. In Vitro Substrate Activity in Activatable Antibodies

This Example demonstrates the in vitro activity of substrates of thedisclosure when they are incorporated into activatable antibodies.

Several substrates identified in these studies were inserted intoProbodies having the 3954 mask and C225v5 variant of cetuximab, which isdescribed in PCT Publication No. WO 2013/163631), which is incorporatedherein by reference in its entirety.

The ability of substrates in the resultant activatable antibodies to becleaved by matriptase-1 or uPA was determined as follows. All proteasedigests were performed in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20pH=7.4. Varying concentrations of active site titrated uPA or matriptasewas combined with a fixed activatable antibody concentration to maintaina substrate to protease ratio of at least 50. Samples were incubated at37° C. for up to 20 h. To stop the reaction, 5 μl of the digest wasadded to 7 μl of HT Protein Express Sample Buffer (Caliper LifeSciences)containing 20 mM 2-Mercaptoethanol for 10 minutes at 95° C. After heatdenaturation, 32 μl of ddH₂O was added and samples analyzed on a LabChipGXII per manufacturer's instructions. The LabChip GXII software was usedto quantify light chain peak area. Product conversion was calculated byplugging the light chain peak areas into the following equation: cleavedLC/(cleaved LC+uncleaved LC), LC=light chain. kcat/Km values weredetermined with the following equation

$\frac{k_{cat}}{k_{m}} = {{- {\ln \left( {1 - C} \right)}}/\left( {t^{*}p} \right)}$

where C is product conversion, t is time (s), and p is proteaseconcentration (M), which assumes that the substrate concentration isbelow the K_(m) and in excess of the protease concentration.

Resultant activatable antibodies comprising substrates selected forcleavage by uPA and matriptase had k_(cat)/K_(M) values ranging fromabout 400 to 5,000 M⁻¹s⁻¹ for uPA and from about 3,000 to 100,000 M⁻¹s⁻¹for matriptase (7 substrates tested). Resultant activatable antibodiescomprising substrates selected for cleavage by matriptase hadk_(cat)/K_(M) values ranging from about 6,500 to 100,000 M⁻¹s⁻¹ formatriptase (5 substrates tested).

Example 5. Substrate Stability of Activatable Antibodies In Vivo

This Example demonstrates the in vivo stability of substrates of thedisclosure when they are incorporated into activatable antibodies andinjected into mice.

Activatable antibodies comprising several substrates of the disclosure,produced as described above, were labeled with either AlexaFluor 680 orDyLight 680 using standard NHS ester chemistry. Unreacted dye wasremoved by purification with a Zeba spin desalting column (40 kDa MWCO,ThermoFisher). Protein concentration was determined by A₂₈₀ using anextinction coefficient calculated from protein sequence and a correctionfactor that accounted for dye absorbance.

Three nude mice (Crl:NU-Foxn1nu) received a single IP dose of eachactivatable antibody at 10 mg/kg or 12.5 mg/kg on Day 0. Mice wereeuthanized on day 4 (about 96 h post-dose) by CO₂ asphyxiation and bloodwas collected immediately as plasma-EDTA and stored at −80° C.

Plasma samples were prepared for analysis by capillary electrophoresisas described in the k_(cat)/K_(m) section. Briefly, 5 μl of plasma wasadded to 7 μl Protein Express Sample Buffer with 2-mercaptoethanol.Quantification of circulating stability was identical to quantificationof product conversion.

Of 14 activatable antibodies comprising substrates of the disclosureselected for cleavage by uPA or by matriptase, 13 exhibited less than20% cleavage in the collected plasma samples.

Example 6. Materials and Methods

Reagents and Strains:

Human uPA (catalog no. 1310-SE, Research & Diagnostics Systems, Inc.)was used without modifications. Human matriptase-1 (catalog no. 3946-SE,Research & Diagnostics Systems, Inc.) was used without modifications.Human tPA (catalog no. HTPA-TC, Molecular Innovations) was used withoutmodifications. Human plasmin (catalog no. HCPM-0140, HaematologicTechnologies Inc.) was used without modifications. Anti-EE monoclonalantibody (Covance, Princeton, N.J.) was labeled with Alexa 647 (LifeSciences) and used with no other modifications (named EE647). E. coliMC1061 or MC1061 derived strains (DH10β) were used for all experiments(Casadaban et al., JMB 138 (2):179-207 (1980)). All bacterial growth wasperformed at 37° C. with vigorous shaking in Luria-Bertani broth (LB)supplemented with 34 μg/mL chloramphenicol (cm), unless anotherantibiotic is specified.

Display Platforms:

Display platforms, each engineered to contain an 8-to-12-amino acidsubstrate of the embodiments, were produced and used as described inInternational Publication No. WO 2014/026136, published 13 Feb. 2014,the contents of which are hereby incorporated by reference in theirentirety. The amino acid sequence of the mature (i.e., without a signalpeptide) CYTX-DP-XXXXXXXX display platform (SEQ ID NO: 694) is shown inFIG. 4A. XXXXXXXX indicates the location into which each substrate isinserted. The amino acid sequence of CYTX-DP-XXXXXXXX display platformalso including its signal peptide, i.e., SP-CYTX-DP-XXXXXXXX displayplatform (SEQ ID NO: 695) is shown in FIG. 4B.

CYTX-DP-XXXXXXXX Display Platform: (SEQ ID NO: 694)GQSGQEYMPMEGGSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGHHHHHHHH SP-CYTX-DP-XXXXXXXX DisplayPlatform: (SEQ ID NO: 695)MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSG GSSGQAAAGHHHHHHHH

Substrate Cleavage and Cleavage Kinetics Analysis:

For clone analysis, overnight cultures were subcultured by dilution intofresh medium (1:40) and grown for 1.5-2 hours. The subculture was theninduced with 0.04% arabinose and incubated with shaking at 37° C. for 40minutes to 1 hour. To stop further growth, cells were then incubated onice for 15 minutes to 1 hour. Cell aliquots were harvested and washedwith reaction buffer. Cells were pelleted by centrifugation, thesupernatant removed and the cells resuspended in reaction buffercontaining the enzyme; the reaction mixture was incubated at 37° C. withshaking. To stop the reaction, cells were removed and diluted 10-fold inPBS, pelleted by centrifugation, and resuspended in PBS containinganti-EE647 (20 micrograms per milliliter (also referred to herein asug/ml or μg/ml)). After incubation on ice (up to 1 hour), cells werewashed with PBS and analyzed using an Accuri C6 cell sorter.

For uPA protease cleavage assays, cultures were induced for 40 minutesto 1 hour. The reaction buffer for uPA was 50 mM Tris-HCl, 150 mM NaCl,0.05% Tween20, pH 7.4 (TBST). Assays for uPA hydrolysis were performedafter cleavage with 2 nM-50 nM uPA for 1 hour. Background hydrolysis ofthe regions flanking the substrate site (using, e.g., CYTX-DP-NSUB, adisplay platform in which the “Substrate” is non-cleavable linkerGGGSGGGS (SEQ ID NO: 696)) was measured under each reaction condition toensure that hydrolysis occurred in the designated substrate region.

For matriptase-1 protease cleavage assays, cultures were induced for 40minutes to 1 hour. The reaction buffer for matriptase-1 was 50 mMTris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.4 (TBST). Assays formatriptase-1 hydrolysis were performed after cleavage with 2 nM-50 nMmatriptase-1 for 1 hr. Background hydrolysis of the regions flanking thesubstrate site (using, e.g., CYTX-DP-NSUB) was measured under eachreaction condition to ensure that hydrolysis occurred in the designatedsubstrate region.

For human plasmin cleavage assays, cultures were induced for 40 minutesto 1 hour. The reaction buffer for plasmin was 50 mM Tris-HCl pH 7.5supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA. Assays forplasmin hydrolysis were performed after cleavage with 20-500 pM plasminfor 1 hr. Background hydrolysis of the regions flanking the substratesite (using, e.g., CYTX-DP-NSUB) was measured under each reactioncondition to ensure that hydrolysis occurred in the designated substrateregion.

For tPA protease cleavage assays, cultures were induced for 40 minutesto 1 hour. The reaction buffer for tPA was 50 mM Tris-HCl, 150 mM NaCl,0.05% Tween20, pH 7.4 (TBST). Assays for tPA hydrolysis were performedafter cleavage with 2 nM-50 nM tPA for 1 hour. Background hydrolysis ofthe regions flanking the substrate site (using, e.g., CYTX-DP-NSUB) wasmeasured under each reaction condition to ensure that hydrolysisoccurred in the designated substrate region.

Amino and Carboxyl Terminus Labeling Conditions:

Alexa-647 conjugated anti-EE antibody (EE647) was used for labeling theEE binding affinity ligand on the N-termini of the CYTX-DP-XXXXXXXXdisplay platform. Alexa-647 conjugated anti-His antibody (His647) wasused for labeling the 8His binding affinity ligand on the C-termini ofthe CYTX-DP-XXXXXXXX display platform. For optimum labeling of cellswithout protease reaction, the cells were incubated for 1 hour at 4° C.with EE647 (1 μg/mL) or His647 (2 μg/ml). For the example describedbelow, a 1-hour incubation was used.

Kinetic Data Analysis:

The extent of conversion of cell surface displayed peptide substrateswas measured directly, using flow cytometry to measure changes in meanfluorescence of clonal cell populations upon protease treatment.Specifically, for each sample, conversion was determined by flowcytometry analyses using the relationship

$\begin{matrix}{{Conversion}_{CLiPS} = \frac{{FL}_{-} - {FL}_{+}}{{FL}_{-} - {FL}_{0}}} & \lbrack 1\rbrack\end{matrix}$

where (FL⁻) is the fluorescence after incubating without enzyme, (FL₊)is fluorescence after incubation with enzyme, and (FL₀) is fluorescenceof unlabeled cells. Given that the expected substrate concentrationsthat were used are significantly below the expected K_(M) of thesubstrate for the target protease, the Michaelis-Menten model simplifiesto

$\begin{matrix}{\frac{d\lbrack S\rbrack}{d\; t} \approx {- {{\frac{k_{cat}}{k_{M}}\lbrack S\rbrack}\lbrack E\rbrack}}} & \lbrack 2\rbrack\end{matrix}$

allowing substrate conversion to be expressed as

$\begin{matrix}{{Conversion}_{MM} = {1 - {\exp \left( {{- \frac{k_{cat}}{k_{M}}} \cdot \lbrack E\rbrack \cdot t} \right)}}} & \lbrack 3\rbrack\end{matrix}$

where [S] is the substrate concentration, [E] is enzyme concentrationand t is time. To determine the second order rate constant(k_(cat)/K_(M)), the time dependent conversion for each substrate wasfit to equation [3].

Example 7. Characterization of Substrate Cleavability in the CYTX-DPDisplay Platform

This Example demonstrates the ability of substrates of the embodimentsto be cleaved by matriptase and/or uPA, but not by plasmin and/or tPA.

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics. Clonesencoding substrates were identified by DNA sequencing and subcloned intothe CYTX-DP-XXXXXXXX display platform such that the expressed displayplatform contained the substrate (typically 8 or 12 amino acids) inplace of XXXXXXXX. Individual substrate-displaying clones (148independent substrate-containing display platforms in total) wereassessed for cleavage by matriptase and/or uPA (target proteases, i.e.,the proteases used to select the substrate) and plasmin and/or tPA(off-target protease); turnover was determined by flow cytometry.Fifty-one of the substrates were selected for cleavage by bothmatriptase and uPA (i.e., Matriptase- and uPA-selected Substrates). Thetwenty-eight Matriptase- and uPA-selected Substrates from Pools wereselected from the same pools as substrates comprising amino acidsequences SEQ ID NOs: 308, 314, and 361 as well as from substratescomprising amino acid sequences 369-371, 374-379, and 381-384. Thetwenty-three Matriptase- and uPA-selected Consensus Substrates wereselected from substrates comprising amino acid sequences SEQ ID NOs:307-311, 313-314, and 320-368. Ninety-seven of the substrates wereselected for cleavage by matriptase (i.e., Matriptase-selectedSubstrates). The fifty-two Matriptase-selected Substrates from Poolswere selected from substrates in Tables 8A through 8J and in Tables 9Athrough 9J-3 as well as from substrates comprising amino acid sequencesSEQ ID NOs: 250-267. The forty-five Matriptase-selected ConsensusSubstrates were selected from substrates comprising amino acid sequencesSEQ ID NOs: 163-249.

In this way, the extent of cleavage for each clone could be determinedand the data aggregated to determine a percent of clones that werecleaved by the target protease and not the off-target protease.Background hydrolysis of the regions flanking the substrate site (using,e.g., the CYTX-DP-NSUB display platform) was measured under eachreaction condition to ensure that hydrolysis occurred in the designatedsubstrate region. Results are presented in Table 12.

TABLE 12 Summary statistics of substrate cleavability >20% <20% CleavageCleavage <20% with 50 nM >20% >20% with Cleavage <20% Matriptase-Cleavage Cleavage with 500 pM with Cleavage Discovery Substrate 1 or 50nM with 50 nM 50 nM Plasmin or 500 pM with effort Group uPA uPAMatriptase-1 50 nM tPA Plasmin 50 nM tPA Matriptase- All 100% 78% 75%76% 76% 96% and uPA- Matriptase- (51 of 51) (40 of 51) (38 of 51) (39 of51) (39 of 51) (49 of 51) selected and uPA- Substrates selectedSubstrates tested Substrates 100% 64% 89% 89% 89% 93% from Pools (28 of28) (18 of 28) (25 of 28) (25 of 28) (25 of 28) (26 of 28) Consensus100% 96% 57% 61% 61% 100% Substrates (23 of 23) (22 of 23) (13 of 23)(14 of 23) (14 of 23) (23 of 23) Matriptase- All 86% 41% 67% 70% 82% 85%selected Matriptase- (83 of 97) (40 of 97) (65 of 97) (68 of 97) (80 of97) (82 of 97) Substrates selected Substrates Substrates 81% 35% 62% 81%94% 87% from Pools (42 of 52) (18 of 52) (32 of 52) (42 of 52) (49 of52) (45 of 52) Consensus 91% 49% 73% 58 69% 82% Substrates (41 of 45)(22 of 45) (33 of 45) (26 of 45) (31 of 45) (37 of 45) Combined Total91% 54% 70% 72% 80% 89% substrates (134 of 148) (80 of 148) (103 of 148)(107 of 148) (119 of 148) (131 of 148) selected for cleavage byMatriptase and/or uPA

Table 12 depicts the percentage of Matriptase- and uPA-selectedSubstrates or Matriptase-selected Substrates tested in the CYTX-DPdisplay platform (a) that exhibited greater than 20% cleavage whenincubated with 50 nM human uPA (catalog no. 1310-SE, Research &Diagnostics Systems, Inc.) used without modifications for 1 hour at 37°C. in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.4 (TBST) (>20%cleavage with 50 nM uPA); (b) that exhibited greater than 20% cleavagewhen incubated with 50 nM human matriptase-1 (catalog no. 3946-SE,Research & Diagnostics Systems, Inc.) used without modifications for 1hour at 37° C. in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.4(TBST) (>20% cleavage with 50 nM matriptase-1); (c) that exhibited lessthan 20% cleavage when incubated with 500 pM human plasmin (catalog no.HCPM-0140, Haematologic Technologies, Inc.) used without modificationsfor 1 hour at 37° C. in 50 mM Tris-HCl pH 7.5 supplemented with 100 mMNaCl, 0.01% Tween20 and 1 mM EDTA (<20% cleavage with 500 pM plasmin);and (d) that exhibited less than 20% cleavage when incubated with 50 nMhuman tPA (catalog no. HTPA-TC, Molecular Innovations) used withoutmodifications for 1 hour at 37° C. in 50 mM Tris-HCl, 150 mM NaCl, 0.05%Tween20, pH 7.4 (TBST) (<20% cleavage with 50 nM tPA).

Example 8. Characterization of Substrate Cleavage Kinetics in theCYTX-DP Display Platform

This Example demonstrates the cleavage kinetics of various substrates ofthe embodiments.

The use of multi-copy substrate display on whole cells enabled simpleand direct quantitative characterization of cleavage kinetics. Cloneswere identified by DNA sequencing and subcloned into theCYTX-DP-XXXXXXXX display platform as described herein. Ninety individualsubstrate-displaying clones were assessed for cleavage, and a subset waschosen to assess cleavage kinetics by the clone's target protease. Theextent of conversion for each clone could be determined at severaldifferent protease concentrations and fit to the Michaelis-Menten modeldescribed herein. Background hydrolysis of the regions flanking thesubstrate site (using, e.g., CYTX-DP-NSUB) was measured under eachreaction condition to ensure that hydrolysis occurred in the designatedsubstrate region. Results are presented in Table 13 & Table 14.

TABLE 13 Summary statistics of uPA substrate kinetics Discovery uPAkcat/Km > uPA kcat/Km > uPA kcat/Km > effort 1 × 10E2 1 × 10E3 1 × 10E4Matriptase- and 100% (18 of 18) 100% (18 of 18) 50% (9 of 18)uPA-selected Substrates Matriptase- 100% (16 of 16) 100% (16 of 16)  6%(1 of 16) selected Substrates Combined 100% (34 of 34) 100% (34 of 34) 29% (10 of 34) Substrates Cleaved by Matriptase and/or uPA

TABLE 14 Summary statistics of Matriptase-1 substrate kineticsMatriptase-1 Matriptase-1 Matriptase-1 kcat/Km > kcat/Km > kcat/Km >Discovery effort 1 × 10E2 1 × 10E3 1 × 10E4 Matriptase- and 100% (25 of25) 100% (25 of 25) 16% (4 of 25)  uPA-selected Substrates Matriptase-100% (31 of 31) 100% (31 of 31) 3% (1 of 31) selected SubstratesCombined 100% (56 of 56) 100% (56 of 56) 9% (5 of 56) Substrates Cleavedby Matriptase and/or uPA

Example 9. In Vivo Efficacy and In Situ Activation of ActivatableAntibodies Comprising a Substrate Cleavable by Matriptase and/or uPA

This Example demonstrates that activatable antibodies comprisingsubstrates of the embodiments cleavable by matriptase and/or uPA areefficacious in vivo. This Example also demonstrates that suchactivatable antibodies are activatable in an in situ imaging assay, suchas that described in International Publication No. WO 2014/107559,published 10 Jul. 2014, the contents of which are hereby incorporated byreference in their entirety.

Three activatable antibodies, each comprising a different substrate ofthe embodiments that are cleaved by matriptase and/or uPA, wereadministered at 10 mg/kg to H292 xenograft tumor-bearing (lung cancer)mice on day 0. All three activatable antibodies also comprised themasking moiety comprising the amino acid sequence CISPRGCPDGPYVMY (SEQID NO: 515) and anti-EGFR antibody C225v5 antibody comprising a lightchain (SEQ ID NO: 458) and a heavy chain (SEQ ID NO: 455). Theconfiguration of the light chain of the activatable antibody was maskingmoiety—substrate—light chain of C225v5.

Mice were retro-orbitally bled on day 4 (about 96 hours post-dose).Blood was collected immediately as plasma-EDTA and stored at −80° C. Thethree activatable antibodies were purified from plasma by anti-human IgGimmunoprecipitation using magnetic beads. To analyze by capillaryelectrophoresis, 5 μl of eluted IgG was added to 7 μl Protein ExpressSample Buffer (Caliper LifeSciences) containing 20 mM 2-Mercaptoethanolfor 10 minutes at 95° C. After heat denaturation, 32 μl of ddH₂O wasadded and samples analyzed on a LabChip GXII per manufacturer'sinstructions. The LabChip GXII software was used to quantify light chainpeak area. Product conversion was calculated by plugging the light chainpeak areas into the following equation: cleaved LC/(cleaved LC+uncleavedLC), LC=light chain peak area. At day 4, the three activatableantibodies demonstrated mean % activation values ranging from 13% to30%. Mean % activation is calculated as ((product conversion sum of thetest group)*100%)/(number of animals in the test group).

The three activatable antibodies demonstrated tumor growth inhibitionranging from 32% to 59% as measured by mean % Δ inhibition. Mean % Δinhibition is calculated as(mean(C)−mean(C0))−(mean(T)−mean(T0))/(mean(C)−mean(C0))*100%, wherein Tis the current test group value, T0 is the current test group initialvalue, C is the control group value, and C0 is the control group initialvalue. The EGFR antibody cetuximab demonstrated 96% inhibition in thisstudy.

The same three activatable antibodies were submitted to in situ imagingassays of mouse xenograft tumor tissues, using the conditions describedin the examples of WO 2014/107559, ibid. The three activatableantibodies were activated, demonstrating that substrates were cleavedand the released antibodies bound to EGFR on the tumor tissue. Thestaining signals ranged from 15% to 85% of the IHC signal intensity ofcetuximab.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

1. An isolated polypeptide comprising a cleavable moiety (CM), whereinthe CM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 363-267, wherein the cleavable moiety is asubstrate for a protease. 2-7. (canceled)
 8. The isolated polypeptide ofclaim 1, wherein the CM is a substrate for at least a matriptaseprotease or a u-plasminogen activator (uPA) protease.
 9. The isolatedpolypeptide of claim 1, wherein the isolated polypeptide comprises atleast one additional moiety (M) selected from the group consisting of amoiety that is located amino (N) terminally to the CM (M_(N)), a moietythat is located carboxyl (C) terminally to the CM (M_(C)), andcombinations thereof.
 10. The isolated polypeptide of claim 9, whereinthe isolated polypeptide comprises at least one M_(N) and at least oneM_(C).
 11. The isolated polypeptide of claim 9, wherein M_(N) isselected from the group consisting of a masking moiety, an antibody, aprotein, a therapeutic agent, an antineoplastic agent, a toxic agent, adrug, a detectable moiety, a diagnostic agent, and an affinity tag. 12.The isolated polypeptide of claim 9, wherein M_(C) is selected from thegroup consisting of a masking moiety, an antibody, a protein, atherapeutic agent, an antineoplastic agent, a toxic agent, a drug, adetectable moiety, a diagnostic agent, and an affinity tag.
 13. Theisolated polypeptide of claim 1, wherein the polypeptide comprises anantibody or antigen binding fragment thereof (AB) that binds a target.14. The isolated polypeptide of claim 13, wherein the CM is a substratefor a protease that is co-localized in a tissue with the target.
 15. Theisolated polypeptide of claim 13, wherein the antigen binding fragmentthereof is selected from the group consisting of a Fab fragment, aF(ab′)₂ fragment, a scFv, a scAb, a dAb, a single domain heavy chainantibody, and a single domain light chain antibody.
 16. The isolatedpolypeptide of claim 13, wherein the AB is linked to the CM.
 17. Theisolated polypeptide of claim 16, wherein the AB is linked directly tothe CM.
 18. The isolated polypeptide of claim 16, wherein the AB islinked to the CM via a linking peptide.
 19. The isolated polypeptide ofclaim 13, wherein the isolated polypeptide comprises a masking moiety(MM), wherein the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB for binding to the target.
 20. The isolatedpolypeptide of claim 19, wherein the MM is a polypeptide of no more than40 amino acids in length.
 21. The isolated polypeptide of claim 19,wherein the MM is linked to the CM such that the isolated polypeptide inan uncleaved state comprises the structural arrangement from N-terminusto C-terminus as follows: MM-CM-AB or AB-CM-MM.
 22. The isolatedpolypeptide of claim 21, wherein the isolated polypeptide comprises alinking peptide between the MM and the CM.
 23. The isolated polypeptideof claim 21, wherein the isolated polypeptide comprises a linkingpeptide between the CM and the AB.
 24. The isolated polypeptide of claim21, wherein the isolated polypeptide comprises a first linking peptide(LP1) and a second linking peptide (LP2), and wherein the isolatedpolypeptide has the structural arrangement from N-terminus to C-terminusas follows in the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.25. The isolated polypeptide of claim 24, wherein the two linkingpeptides need not be identical to each other.
 26. The isolatedpolypeptide of claim 24, wherein each of LP1 and LP2 is a peptide ofabout 1 to 20 amino acids in length.
 27. The isolated polypeptide ofclaim 19, wherein the amino acid sequence of the MM is different fromthat of the target.
 28. The isolated polypeptide of claim 19, whereinthe MM does not interfere or compete with the AB for binding to thetarget in a cleaved state.
 29. The isolated polypeptide of claim 13comprising a T cell-engaging scFv linked to the AB.
 30. The isolatedpolypeptide of claim 29, wherein the T cell-engaging scFv comprises amasking moiety.
 31. The isolated polypeptide of claim 19 comprising a Tcell-engaging scFv linked to the AB.
 32. The isolated polypeptide ofclaim 31, wherein the T cell-engaging scFv comprises a masking moiety.33. The isolated polypeptide of claim 13 conjugated to an agent.
 34. Theisolated polypeptide of claim 33, wherein the agent is a toxin orfragment thereof.
 35. The isolated polypeptide of claim 33, wherein theagent is selected from the group consisting of a dolastatin or aderivative thereof, an auristatin or a derivative thereof, amaytansinoid or a derivative thereof, a duocarmycin or a derivativethereof, and a calicheamicin or a derivative thereof.
 36. The isolatedpolypeptide of claim 33, wherein the agent is a detectable moiety. 37.The isolated polypeptide of claim 33, wherein the agent is conjugated tothe polypeptide via a linker.
 38. The isolated polypeptide of claim 37,wherein the linker is a cleavable linker.
 39. The isolated polypeptideof claim 19, wherein the AB is conjugated to an agent.
 40. The isolatedpolypeptide of claim 39, wherein the agent is a toxin or fragmentthereof.
 41. The isolated polypeptide of claim 39, wherein the agent isselected from the group consisting of a dolastatin or a derivativethereof, an auristatin or a derivative thereof, a maytansinoid or aderivative thereof, a duocarmycin or a derivative thereof, and acalicheamicin or derivative thereof.
 42. The isolated polypeptide ofclaim 39, wherein the agent is auristatin E or a derivative thereof. 43.The isolated polypeptide of claim 39, wherein the agent is monomethylauristatin E (MMAE).
 44. The isolated polypeptide of claim 39, whereinthe agent is monomethyl auristatin D (MMAD).
 45. The isolatedpolypeptide of claim 39, wherein the agent is DM1 or DM4.
 46. Theisolated polypeptide of claim 39, wherein the agent is conjugated to theAB via a linker.
 47. The isolated polypeptide of claim 46, wherein thelinker is a cleavable linker.
 48. The isolated polypeptide of claim 13comprising a detectable moiety.
 49. The isolated polypeptide of claim48, wherein the detectable moiety is a diagnostic agent. 50-57.(canceled)